FIELD OF THE INVENTION

The present invention relates to combination therapy involving two or more Fc region-containing antigen-binding polypeptides, such as antibodies, wherein the polypeptides have been modified such that hetero-oligomerization between the polypeptides is strongly favored over homo-oligomerization when the polypeptides are bound to their corresponding target antigens. The invention also relates to polypeptides, use of such polypeptides, compositions, kits and devices suitable for use in the combination therapy of the invention.

BACKGROUND OF THE INVENTION

Antibodies are highly effective molecules which can have effects on target cells via various mechanisms. In some instances, the mere binding of an antibody to a target antigen on a cell surface can have an antagonistic or agonistic effect on the target antigen and thus on the target cell. Alternatively, or in addition, the effect of an antibody on a target cell is achieved through the ability of antibodies to induce effector functions, typically Fc-mediated effector functions, such as complement- dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cell-mediated phagocytosis (ADCP).

ADCC and ADCP are initiated by binding of the IgG Fc region to Fey receptors on effector cells. W02012/130831 discloses Fc region-containing polypeptides that have altered ADCC function as a consequence of one or more amino acid substitutions in the Fc region of the polypeptide.

CDC is initiated by binding of Clq to the Fc regions of antibodies. Clq is a multimeric protein consisting of six globular binding heads attached to a stalk. The individual globular binding heads have low affinity for IgG, and Clq must gain avidity by binding multiple IgGl molecules on a cell surface to trigger the classical complement pathway. IgG hexamerization upon target binding on the cell surface has been shown to support avid Clq binding. The hexamerization is mediated through intermolecular non-covalent Fc-Fc interactions. Fc-Fc interactions can be enhanced by point mutations in the CH3 domain, including E345R and E430G (see, e.g. WO2013/004842 and W02014/108198). WO2017/093447 is directed to antibodies that bind a death receptor comprising an intracellular death domain. The application discloses that a K439E mutation in the Fc region of an antibody results in Fc-Fc repulsion, and thus weak Fc-Fc interactions between two antibody molecules having said mutation. This effect could be neutralized by introducing a S440K mutation in the other antibody molecule, leading to a restoration of the Fc-Fc interactions, see also Diebolder et al. (2014) Science 343: 126.

While antibody therapy is often highly efficacious, antibody target antigens are often not uniquely expressed in diseased cells or tissue, but are also found in other, healthy, cells or tissue. Thus, antibody therapy may lack selectivity for the target tissue and non-diseased tissue may be affected by the antibody treatment resulting in toxicity.

There is therefore a need for improved antibody treatment, in particular treatment with improved selectivity.

Accordingly, it is an object of the present invention to provide for a method of treating a disease by increasing the selectivity of polypeptides or antibodies. It is yet a further object of the present invention to provide for a method of treating a disease by providing for a first and a second polypeptide which have no single agent activity, but only show activity when bound together on the same target cell or tissue. Thus, it is an object of the present invention to provide for a method of treating a disease by administering a first polypeptide capable of binding to a first antigen and a second polypeptide capable of binding to a second antigen, wherein the first and second polypeptide has no effect, or only little effect, on tissues or target organs expressing either the first or second antigen, while providing effective treatment on tissues or target organs expressing both the first and second antigen. It is a further object of the present invention to provide for a method of treating a disease by administering to a subject a first polypeptide and a second polypeptide which have been modified to prevent homo-oligomerization (self-oligomerization) while favoring hetero- oligomerization. It is another object of the present invention to provide for polypeptides, which may be used in such method of treatment, i.e. polypeptides having at least one self-oligomerization inhibiting mutation. It is yet another object of the present invention to provide for a first polypeptide having a self- oligomerization inhibiting mutation and a second polypeptide having a self- oligomerization inhibiting mutation, where the self-oligomerization inhibiting mutations in the first and second polypeptide are complementary, thereby allowing for hetero-oligomerization of the first and second polypeptide when bound to a target cell. SUMMARY OF THE INVENTION

The present invention provides methods, polypeptides and compositions which can be used to improve the selectivity of an antibody treatment for desired target cell populations.

The methods or uses of the invention relate to a treatment with two antibodies (or antibody-like polypeptides), wherein the two antibodies bind two different target antigens and wherein the Fc regions of the antibodies have been modified such that hetero-oligomerization of the two antibodies is strongly favored over homo-oligomerization. As a result of these modifications, more antibody oligomerization will occur on cells that express both antigen targets (allowing efficient (hetero)oligomerization of the two antibodies), than on cells that only express one of the targets (resulting in inefficient or no (homo)oligomerization). As oligomerization generally enhances the efficacy of antibodies, the antibody combination treatment will be more efficacious against cells that co-express the targets than against cells that only express one of the targets. Thus, the antibody combination treatment has an improved selectivity for cells or tissue expressing both target antigens. Accordingly, by selecting two antigens that are co-expressed in a desired target cell population, but not, or less, co-expressed in cell populations that should not be targeted, a combined antibody treatment can be designed which will have a selective effect against the desired target cell populations.

It is contemplated that the increased efficacy will not only be obtained when the two target antigens are co-expressed on the same cell, but also in other situations where the target cells are in close proximity. Furthermore, besides classical antibodies, also antibody-like polypeptides can be used provided that they comprise an Fc region and an antigen-binding region.

Accordingly, in a first aspect, the invention relates to a method of treating a disease or disorder comprising administering to a subject in need thereof: a first polypeptide comprising a first Fc region of a human IgG and a first antigen-binding region capable of binding to a first antigen, in combination with a second polypeptide comprising a second Fc region of a human IgG and a second antigen-binding region capable of binding to a second antigen, wherein

a) said first polypeptide comprises an I253G mutation of an amino acid position corresponding to 1253 in human IgGl and said second polypeptide comprises an H310R mutation of an amino acid position corresponding to H310 in human IgGl, or vice versa, or

said first polypeptide comprises an I253K or I253R mutation of an amino acid position corresponding to 1253 in human IgGl and said second polypeptide comprises an H310D mutation of an amino acid position corresponding to H310 in human IgGl, or vice versa,

and/or

b) said first polypeptide comprises a Y436N, Y436K, Y436Q or Y436R mutation of an amino acid position corresponding to Y436 in human IgGl and said second polypeptide comprises a Q438R, Q438K, Q438H, Q438G or Q438N mutation of an amino acid position corresponding to Q438 in human IgGl, or vice versa, or

said first polypeptide comprises a Y436N or Y436Q mutation of an amino acid position corresponding to Y436 in human IgGl and said second polypeptide comprises a Y436K or Y436R mutation of an amino acid position corresponding to Y436 in human IgGl, or vice versa,

or

said first polypeptide comprises a Q438R, Q438K or Q438H mutation of an amino acid position corresponding to Q438 in human IgGl and said second polypeptide comprises a Q438N or Q438G mutation of an amino acid position corresponding to Q438 in human IgGl, or vice versa,

and/or

c) said first polypeptide comprises a K439F, K439I, K439Y, K439T, K439V, K439W mutation of an amino acid position corresponding to K439 in human IgGl and said second polypeptide comprises an S440K mutation of an amino acid position corresponding to S440 in human IgGl, or vice versa,

wherein the amino acid positions correspond to human IgGl according to EU numbering.

In one embodiment of the method of the invention, said first polypeptide comprises a Y436N or Y436K mutation of an amino acid position corresponding to Q436 in human IgGl and said second polypeptide comprises a Q438N or Q438R mutation of an amino acid position corresponding to Q438 in human IgGl, or vice versa.

In one aspect of the invention said first polypeptide comprises a F436N, F436K, F436Q or F436R mutation of an amino acid position corresponding to F436 in human IgG3 and said second polypeptide comprises a Q438R, Q438K, Q438H, Q438G or Q438N mutation of an amino acid position corresponding to Q438 in human IgGl, or vice versa,

or

said first polypeptide comprises a F436N or F436Q mutation of an amino acid position corresponding to F436 in human IgG3 and said second polypeptide comprises a F436K or F436R mutation of an amino acid position corresponding to F436 in human IgG3, or vice versa.

In a further aspect, the invention relates to a first polypeptide, comprising a first Fc region of a human IgG and a first antigen-binding region capable of binding to a first antigen, for use as a medicament in combination with a second polypeptide, comprising a second Fc region of a human IgG and a second antigen-binding region capable of binding to a second antigen, wherein

a) said first polypeptide comprises an I253G mutation of an amino acid position corresponding to 1253 in human IgGl and said second polypeptide comprises an H310R mutation of an amino acid position corresponding to H310 in human IgGl, or vice versa,

or

said first polypeptide comprises an I253K or I253R mutation of an amino acid position corresponding to 1253 in human IgGl and said second polypeptide comprises an H310D mutation of an amino acid position corresponding to H310 in human IgGl, or vice versa,

and/or

b) said first polypeptide comprises a Y436N, Y436K, Y436Q or Y436R mutation of an amino acid position corresponding to Y436 in human IgGl and said second polypeptide comprises a Q438R, Q438K, Q438H, Q438G or Q438N mutation of an amino acid position corresponding to Q438 in human IgGl, or vice versa, or

said first polypeptide comprises a Y436N or Y436Q mutation of an amino acid position corresponding to Y436 in human IgGl and said second polypeptide comprises a Y436K or Y436R mutation of an amino acid position corresponding to Y436 in human IgGl, or vice versa,

or

said first polypeptide comprises a Q438R, Q438K or Q438H mutation of an amino acid position corresponding to Q438 in human IgGl and said second polypeptide comprises a Q438N or Q438G mutation of an amino acid position corresponding to Q438 in human IgGl, or vice versa,

and/or

c) said first polypeptide comprises a K439F, K439I, K439Y, K439T, K439V, K439W mutation of an amino acid position corresponding to K439 in human IgGl and said second polypeptide comprises an S440K mutation of an amino acid position corresponding to S440 in human IgGl, or vice versa,

wherein the amino acid positions correspond to human IgGl according to EU numbering.

In a further aspect, the invention relates to a polypeptide comprising an Fc region of a human IgG and an antigen-binding region capable of binding to an antigen, wherein said polypeptide comprises

a) a I253G, I253K or I253R mutation of an amino acid position corresponding to 1253 in human IgGl,

or

a H310R or H310D or mutation of an amino acid position corresponding to H310 in human IgGl,

and/or

b) a Y436N, Y436K, Y436Q or Y436R mutation of an amino acid position corresponding to Y436 in human IgGl,

or

a Q438R, Q438K, Q438H, Q438G or Q438N mutation of an amino acid position corresponding to Q438 in human IgGl,

and/or

c) a K439F, K439I, K439Y, K439T, K439V, K439W mutation of an amino acid position corresponding to K439 in human IgGl,

or

an S440K mutation of an amino acid position corresponding to S440 in human IgGl,

wherein the amino acid positions correspond to human IgGl according to EU numbering,

with the proviso that if the polypeptide comprises said S440K mutation, then at least one of the other mutations specified in options a) and b) is also present. In another aspect, the invention relates to compositions comprising one or more polypeptides of the invention as defined herein.

In further aspects, the invention relates to pharmaceutical compositions comprising one or more polypeptides of the invention as defined herein.

In a further aspect, the invention relates to a kit comprising a first container comprising a first polypeptide suitable for use in the invention as defined herein and a second container comprising a second polypeptide suitable for use in the invention as defined herein.

In an even further aspect, the invention relates to a device, such as a dual chamber syringe, comprising a first compartment comprising a first polypeptide suitable for use in the invention as defined herein and a second compartment comprising a second polypeptide suitable for use in the invention as defined herein.

These and other aspects of the invention, particularly various uses and therapeutic applications for the polypeptide or antibody, are described in further detail below.

Brief Description of the Drawings

Figure 1 shows the amino acid sequence alignment of the human IgGlm(a), IgGlm(f), IgG2, IgG3 and IgG4 Fc backbones with the EU based (IgGl-specific) numbering scheme (Edelman et al. 1969 Proc Natl Acad Sci USA 63, 78-85).

Figure 2 shows the results of a CDC assay testing IgGl-Campath-E430G antibody variants with the indicated 1253 or H310 mutations for the effect of manipulating Fc- Fc interactions and CDC efficacy on Wien 133 cells. Wien 133 cells were incubated with concentration series of the single antibody variants and all possible antibody combinations of 1253 + H310 mutant pairs in the presence of 5% pooled normal human serum (NHS). CDC efficacy is presented as the half maximal effective antibody concentration (EC50) in ng/ml_, as determined by the percentage of TO- PRO-3 iodide-positive cells. Maximal cell lysis with an undefinable low EC50 value is indicated as < 15 ng/ml_.

Figure 3 shows the results of a CDC assay testing IgGl-Campath-E430G antibody variants with the indicated Y436 or Q438 mutations for the effect of manipulating Fc- Fc interactions and CDC efficacy on Wien 133 cells. Wien 133 cells were incubated with concentration series of the single antibody variants and all possible antibody combinations of Y436 + Q438 mutant pairs in the presence of 5% pooled NHS. CDC efficacy is presented as the half maximal effective antibody concentration (EC50) in ng/mL, as determined by the percentage of TO-PRO-3 iodide-positive cells. Maximal cell lysis with an undefinable low EC50 value is indicated as < 15 ng/mL.

Figure 4 shows the results of a CDC assay testing IgGl-Campath-E430G antibody variants with the indicated K439 or S440 mutations for the effect of manipulating Fc- Fc interactions and CDC efficacy on Wien 133 cells. Wien 133 cells were incubated with concentration series of the single antibody variants and all possible antibody combinations of K439 + S440 mutant pairs in the presence of 5% pooled NHS. CDC efficacy is presented as the half maximal effective antibody concentration (EC50) in ng/mL, as determined by the percentage of TO-PRO-3 iodide-positive cells. Maximal cell lysis with an undefinable low EC50 value is indicated as < 15 ng/mL.

Figure 5 shows the effect of Fc-Fc inhibiting mutations I253G and H310R (A), I253K and H310D (B), and I253R and H310D (C) on the CDC efficacy of IgGl-Campath- E430G. Wien 133 cells were incubated with concentration series of the indicated IgGl-Campath-E430G antibody variants containing a single Fc-Fc inhibiting mutation (single mAh) and the combinations thereof (mAh mixture) in the presence of 20% pooled NHS. CDC efficacy is presented as the percentage lysis determined by the percentage Pi-positive cells. The IgG-bl2 antibody against HIV gpl20 was used as a non-binding control antibody.

Figure 6 shows the effect of combining Fc-Fc inhibiting mutations on the CDC efficacy of IgGl-Campath-E430G. Wien 133 cells were incubated with concentration series of the indicated IgGl-Campath-E430G antibody variants containing one or two Fc-Fc inhibition mutations in single antibodies (single mAh) and in combinations thereof (mAh mixture) in the presence of 20% pooled NHS. CDC efficacy is presented as the percentage lysis determined by the percentage Pi-positive cells. The IgG-bl2 antibody against HIV gpl20 was used as a non-binding control antibody.

Figure 7 shows FcRn binding of anti-CD52 IgGl-CAMPATH-lH antibodies with self- oligomerization inhibiting substitutions. Binding to human FcRn is shown of antibody variants of anti-CD52 IgGl-CAMPATH-lH-E430G-K439E and anti-CD52 IgGl- CAMPATH-1H-E430G-S440K with the self-oligomerization inhibiting substitutions I253G, I253K, I253R, H310D, H310R, Y436N, Y436K, Q438N and/or Q438R using a 40 pg/ml antibody concentration at (A) pH 7.4 and (B) pH 6.0. An FcRn ELISA was performed with 5 pg/mL coated recombinant extracellular domain of human FcRn (FcRnECDHis-B2M-BIO) and antibody dilution series. The amount of bound antibodies was determined with an HRP-conjugated goat anti-human IgGl antibody and the chemiluminescent substrate ABTS. Absorbance was measured at 405 nm. Figure 8 shows FcyR binding of IgG 1-CAM PATH- 1H variants with Fc-Fc enhancing mutation E430G and self-oligomerization inhibiting substitutions. Binding of immobilized IgGl-CAMPATH-lH-E430G variants with self-oligomerization inhibiting substitutions K439E, S440K, Y436K, Y436N, Q438N and Q438R to dimeric His- tagged biotinylated ECDs of (A) FcyRIIA allotype 131H, (B) FcyRIIA allotype 131R, (C) FcyRIIB, (D) FcyRIIIA allotype 158V and (E) FcyRIIIA allotype 158F as tested in ELISA assays. Binding is presented for 20 pg/mL antibody samples relative to no antibody control (background) and binding to IgGl-CAMPATH-lH-E430G (100%). Detection was performed using Streptavidin-polyHRP and ABTS.

Figure 9 shows CDC efficacy of single agent and combined anti-CD52 IgGl- CAMPATH-1H-E430G-K439E and anti-CD52 IgGl-CAMPATH-lH-E430G-S440K antibodies harboring self-oligomerization inhibiting mutations. Wien 133 cells were incubated with antibody concentration series in the presence of 20% NHS. CDC efficacy is presented as (A) the AUC normalized to non-binding control antibody IgGl-bl2 (0%) and IgGl-CAMPATH-lH-E430G (100%) and (B) percentage lysis determined by the percentage Pi-positive cells at an antibody concentration of 40 pg/ml.

Figure 10 shows CDC efficacy of single agent and combined anti-CD20 IgGl-llB8- E430G-K439E and anti-CD20 IgGl-llB8-E430G-S440K antibodies harboring additional self-oligomerization inhibiting mutations. Wien 133 cells were incubated with antibody concentration series in the presence of 20% NHS. CDC efficacy is presented as the AUC normalized to non-binding control antibody IgGl-bl2 (0%) and the mixture of IgGl-CAMPATH-lH-E430G (CAMP-E430G) + IgGl-llB8-E430G ( 100%).

Figure 11 shows CDC efficacy of single agent and combined anti-CD52 IgGl- CAMPATH-1H-E430G-K439E and anti-CD20 IgGl-llB8-E430G-S440K antibodies harboring self-oligomerization inhibiting mutations. Wien 133 cells were incubated with antibody concentration series in the presence of 20% NHS. CDC efficacy is presented as the AUC normalized to non-binding control antibody IgGl-bl2 (0%) and the mixture of IgGl-CAMPATH-lH-E430G + IgGl-llB8-E430G (100%).

Figure 12 shows CDC efficacy of single agent and combined variants of anti-CD52 IgGl-CAMPATH-lH and anti-CD20 IgGl-llB8 antibodies harboring different Fc-Fc interaction enhancing mutations. (A, B) Wien 133 cells were incubated with antibody concentration series in the presence of 20% NHS. CDC efficacy is presented as the AUC normalized to non-binding control antibody IgGl-bl2 (0%) and the mixture of IgG 1-CAM PATH- 1H-E430G + IgGl-llB8-E430G (100%).

Figure 13 shows selectivity of CDC activity by mixed antibody subclass variants (IgGl, IgG2 and hinge-stabilized IgG4) of anti-CD52 CAMPATH-1H-E430G-K439E with additional self-oligomerization inhibiting mutations + anti-CD20 11B8-E430G- S440K with additional self-oligomerization inhibiting mutations. Wien 133 cells were incubated with antibody concentration series in the presence of 20% NHS. CDC efficacy is presented as the normalized AUC of the percentage Pi-positive cells. Normalization was performed to non-binding control antibody IgGl-bl2 (0%) and the mixture of IgGl-CAMPATH-lH-E430G + IgGl-llB8-E430G (100%).

Figure 14 shows the effect of introducing FcyR-binding inhibiting mutation G237A in IgGl-CAMPATH-lH and IgGl-llB8 variants with Fc-Fc interaction enhancing and self-oligomerization inhibiting mutations on FcyR binding and CDC activity. Binding of immobilized IgG 1-CAM PATH- 1H and IgGl-llB8 variants with self-oligomerization inhibiting mutations K439E or S440K to dimeric His-tagged biotinylated ECDs of (A) FcyRIIA allotype 131H, (B) FcyRIIA allotype 131R, (C) FcyRIIB, (D) FcyRIIIA allotype 158F and (E) FcyRIIIA allotype 158V as tested in ELISA assays. Binding is presented as the absorbance at 405 nm wavelength for 20 pg/mL antibody samples. Detection was performed using Streptavidin-polyHRP and ABTS. (F, G) Wien 133 cells were incubated with antibody concentration series in the presence of 20% NHS. CDC efficacy is presented as the normalized AUC of the percentage Pi-positive cells. Normalization was performed to non-binding control antibody IgGl-bl2 (0%) and IgGl-CAMPATH-lH-E430G (100%; F) or to non-binding control antibody IgGl-bl2 (0%) and a mixture of IgGl-CAMPATH-lH-E430G + IgGl-llB8-E430G (100%; G). Figure 15 shows the selectivity of CDC activity by mixed antibody variants of anti- CD52 CAM PATH- 1H-E430G-K439E and anti-CD20 11B8-E430G-S440K with or without self-oligomerization inhibiting mutations, FcyR-binding inhibiting mutation G237A, and/or Clq-binding-enhancing mutations E333S or K326W-E333S. Wien 133 cells were incubated with antibody concentration series in the presence of 20% NHS. CDC efficacy is presented as the normalized AUC of the percentage Pi-positive cells. Normalization was performed to non-binding control antibody IgGl-bl2 (0%) and the mixture of IgGl-CAMPATH-lH-E430G + IgGl-llB8-E430G (100%).

Figure 16 shows CDC efficacy of single agent and combined anti-CD37 IgGl-CD37- 37-3-E430G antibody variants harboring self-oligomerization inhibiting mutations. Raji cells were incubated with antibody concentration series in the presence of 20% NHS. CDC efficacy is presented as the AUC norma lized to non-binding control antibody IgGl-b l2 (0%) and IgG 1-CAM PATH- 1 H-E430G + IgG l-CD37-37-3-E430G ( 100%).

Figure 17 shows CDC efficacy of single agent and combined anti-CD37 IgG l-CD37- 37-3-E430G and IgG l- l lB8-E430G antibody va riants harboring self-oligomerization inhibiting mutations. Raji cells were incubated with antibody concentration series in the presence of 20% NHS. CDC efficacy is presented as the AUC normalized to non- binding control antibody IgGl-b l2 (0%) and IgG l-CAM PATH- lH-E430G + IgG l- CD37-37-3-E430G ( 100%).

Figure 18 shows CDC efficacy of single agent and combined anti-CD52 IgG l- CAMPATH- 1H-E430G anti-CD37 IgG l-CD37-37-3-E430G antibody variants harboring self-oligomerization inhibiting mutations. Raji cells were incubated with antibody concentration series in the presence of 20% NHS. CDC efficacy is presented as the AUC normalized to non-binding control antibody IgGl-b l2 (0%) and IgG l-CAMPATH- 1H-E430G + IgGl-CD37-37-3-E430G ( 100%).

Figure 19 shows cytotoxicity of anti-DR5 antibody variants of IgGl-DR5-01-G56T- E430G and IgGl-DR5-05-E430G harboring self-oligomerization in hibiting mutations. BxPC-3 cells were incubated with antibody concentration series in the presence of purified human Clq (2.9 pg/mL final concentration).. Cytotoxicity is presented as the cell viability at 20 pg/ml antibody concentration. The percentage viable cells was calculated using the following formula : % viable cells = [(luminescence antibody sample - luminescence staurosporine sample)/(luminescence no antibody sample - luminescence staurosporine sample)]* 100%.

Figure 20 shows CDC efficacy of single agent and combined anti-CD37 IgGl-7D8- E430G antibody variants harboring self-oligomerization inhibiting mutations. Raji cells were incubated with antibody concentration series in the presence of 20% NHS. CDC efficacy is presented as the AUC norma lized to non-bind ing control antibody IgG l-bl2 (0%) and IgGl-CAM PATH-l H-E430G + IgG l-7D8-E430G ( 100%).

Figure 21 shows CDC efficacy by anti-CD52 IgGl-CAMPATH- lH-E430G and anti- CD20 IgGl- l lB8-E430G antibody va riants harboring self-oligomerization inhibiting mutations, as single agents or mixed in different ratios. Wien 133 cells were incubated with antibody concentration series in the presence of 20% NHS. CDC efficacy is presented as the percentage of cell lysis calculated from the number of PI- positive cells. (A) CDC efficacy of single agents IgG l-CAM PATH-lH-E430G-K439E- Q438N and IgGl-llB8-E430G-S440K-Y436K and mixtures thereof. (B) CDC efficacy of single agents IgGl-CAMPATH-lH-E430G-K439E-Q438N and IgGl-llB8-E430G- S440K-Q438R and mixtures thereof.

Figure 22 shows CDC efficacy of single agent and combined anti-CD52 IgGl- CAMPATH-1H-E430G and non-antigen-binding IgGl-bl2-E430G antibody variants harboring self-oligomerization inhibiting mutations. Wien 133 cells were incubated with antibody concentration series in the presence of 20% NHS. CDC efficacy is presented as the AUC normalized to non-binding control antibody IgGl-bl2 (0%) and IgG 1-CAM PATH- 1H-E430G (100%). (A) CDC and (B) maximal cell lysis induced by single agent antibody variants harboring mutation E430G in combination with either mutation K439E or S440K and mixtures thereof. (C) CDC and (D) maximal cell lysis induced by antibody variants harboring the E430G, K439E, and Y436N mutations mixed with IgGl-CAMPATH-lH or IgGl-bl2 antibody variants harboring complementary mutations, compared to their single agent control reactions. (E) CDC and (F) maximal cell lysis induced by antibody variants harboring the E430G, K439E, and Q438N mutations mixed with IgGl-CAMPATH-lH or IgGl-bl2 antibody variants harboring complementary mutations, compared to their single agent control reactions.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

The term "polypeptide comprising an Fc-region of an IgG and an antigen- binding region" refers in the context of the present invention to a polypeptide which comprises an Fc-region of an immunoglobulin of the IgG isotype and a binding region which is a capable of binding to an antigen, which can be any type of molecule, such as a polypeptide, e.g. present on a cell, bacterium, or virion. The Fc-region of an immunoglobulin is defined as the fragment of an antibody which would be typically generated after digestion of an antibody with papain (which is known for someone skilled in the art) which includes the two CH2-CH3 regions of an immunoglobulin and a connecting region, e.g. a hinge region. Thus, the term "Fc-region of an IgG" means in the context of the present invention that a connecting region, e.g. hinge region, and the CH2 and CH3 region of an immunoglobulin are present. The constant domain of an antibody heavy chain defines the antibody isotype, which can e.g. be IgGl, IgG2, IgG3 or IgG4. The Fc-region mediates the effector functions of antibodies with cell surface receptors called Fc receptors and proteins of the complement system. The polypeptide comprising an Fc-domain of an IgG and an antigen-binding region may be an antibody, like a chimeric, humanized, or human antibody or a heavy chain only antibody or a ScFv-Fc-fusion, or an Fc-fusion-protein. The polypeptide is not limited to human origin but can be of any origin, such as e.g. mouse, rat, rabbit or cynomolgus origin.

The term "immunoglobulin" or "Ig" refers to a class of structurally related glycoproteins consisting of two pairs of polypeptide chains, one pair of light (L) low molecular weight chains and one pair of heavy (H) high molecular weight chains, all four potentially inter-connected by disulfide bonds. "IgG" refers to an immunoglobulin G. The structure of immunoglobulins has been well characterized. See for instance Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed . Raven Press, N.Y. ( 1989)). Briefly, each heavy chain typically is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region typically is comprised of three domains, CHI, CH2, and CH3. The heavy chains are inter-connected via disulfide bonds in the so-called "hinge region". Each light chain typically is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region typically is comprised of one domain, CL. The VH and VL regions may be further subdivided into regions of hypervariability (or hypervariable regions which may be hypervariable in sequence and/or form of structurally defined loops), also termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs). Each VH and VL is typically composed of three CDRs and four FRs, arranged from amino-terminus to carboxy- terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (see also Chothia and Lesk J . Mol. Biol. 196, 901 917 (1987)). Unless otherwise stated or contradicted by context, CDR sequences herein are identified according to IMGT rules using DomainGapAlign (Lefranc MP., Nucleic Acids Research 1999;27 :209-212 and Ehrenmann F., Kaas Q. and Lefranc M .-P. Nucleic Acids Res., 38, D301-307 (2010); see also internet http address www.imqt.orq/. Unless otherwise stated or contradicted by context, reference to amino acid positions in the present invention is according to the EU-numbering (Edelman et al., Proc Natl Acad Sci U S A. 1969 May;63(l) :78-85; Rabat et al., Sequences of proteins of immunological interest. 5th Edition - 1991 NIH Publication No. 91-3242).

The term "amino acid corresponding to position..." as used herein refers to an amino acid position number in a human IgGl heavy chain. Corresponding amino acid positions in other immunoglobulins may be found by alignment with human IgGl. Figure 1 gives an alignment of IgGl, IgG2, IgG3 and IgG4 sequences showing which positions in IgG2, IgG3 and IgG4 correspond to which positions in IgGl. Thus, an amino acid or segment in one sequence that "corresponds to" an amino acid or segment in another sequence is one that aligns with the other amino acid or segment using a standard sequence alignment program such as ALIGN, ClustalW or similar, typically at default settings and has at least 50%, at least 80%, at least 90%, or at least 95% identity to a human IgGl heavy chain. It is considered well-known in the art how to align a sequence or segment in a sequence and thereby determine the corresponding position in a sequence to an amino acid position according to the present invention.

The term "hinge region" as used herein is intended to refer to the hinge region of an immunoglobulin heavy chain. Thus, for example the hinge region of a human IgGl antibody corresponds to amino acids 216-230 according to the EU numbering.

The term "CH2 region" or "CH2 domain" as used herein is intended to refer the CH2 region of an immunoglobulin heavy chain. Thus, for example the CH2 region of a human IgGl antibody corresponds to amino acids 231-340 according to the EU numbering. However, the CH2 region may also be any of the other isotypes as described herein.

The term "CH3 region" or "CH3 domain" as used herein is intended to refer the CH3 region of an immunoglobulin heavy chain. Thus, for example the CH3 region of a human IgGl antibody corresponds to amino acids 341-447 according to the EU numbering. However, the CH3 region may also be any of the other isotypes as described herein.

The term "Fc region" or "Fc domain", which may be used interchangeably herein, refers to an antibody region comprising, arranged from amino-terminus to carboxy-terminus, at least a hinge region, a CH2 domain and a CH3 domain. An Fc region of an IgG l antibody can, for example, be generated by digestion of an IgGl antibody with papain.

The term "antibody" (Ab) in the context of the present invention refers to an immunoglobulin molecule, a fragment of an immunoglobulin molecule, or a derivative of either thereof, which has the ability to specifically bind to an antigen. An antibody used in the present invention comprises an Fc-domain of an immunoglobulin and an antigen-binding region. An antibody generally contains a CH2-CH3 region and a connecting region, e.g . a hinge region, e.g. at least an Fc- domain. The variable regions of the heavy and light chains of the immunoglobulin molecule contain a binding domain that interacts with an antigen. An antibody may also be a monospecific or a multispecific antibody, such as a bispecific antibody or similar molecule. The term "bispecific antibody" refers to an antibody having specificities for at least two different, typically non-overlapping, epitopes. Such epitopes may be on the same or different targets. If the epitopes are on different targets, such targets may be on the same cell or different cells or cell types. As indicated above, unless otherwise stated or clearly contradicted by the context, the term antibody herein includes fragments of an antibody which comprise at least a portion of an Fc-region and which retain the ability to specifically bind to the antigen. Such fragments may be provided by any known technique, such as enzymatic cleavage, peptide synthesis and recombinant expression techniques. It has been shown that the antigen-binding function of an antibody may be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "Ab" or "antibody" include, without limitation, monovalent antibodies (described in W02007059782 by Genmab); heavy-chain antibodies, consisting only of two heavy chains and naturally occurring in e.g. camelids (e.g ., Hamers- Casterman ( 1993) Nature 363 :446); ThioMabs (Roche, W02011069104); strand- exchange engineered domain (SEED or Seed-body) which are asymmetric and bispecific antibody-like molecules (Merck, W02007110205); Triomab (Pharma/Fresenius Biotech, Lindhofer et al. 1995 J Immunol 155: 219; W02002020039); FcAAdp (Regeneron, W02010151792), Azymetric Scaffold (Zymeworks/Merck, WO2012/058768); mAb-Fv (Xencor, WO2011/028952), Xmab (Xencor); Dual variable domain immunoglobulin (Abbott, DVD-Ig, U.S. Patent No. 7,612,181); Dual domain double head antibodies (Unilever; Sanofi Aventis, W020100226923); Di-diabody (ImClone/Eli Lilly); Knobs-into-holes antibody formats (Genentech, WO9850431 ); DuoBody (Genmab, WO 2011/131746); Bispecific IgGl and IgG2 (Pfizer/ Rinat, W011143545); DuetMab (Medlmmune, US2014/0348839); Electrostatic steering antibody formats (Amgen, EP1870459 and WO 2009089004; Chugai, US201000155133; Oncomed, W02010129304A2); CrossMAbs (Roche, WO2011117329); LUZ-Y (Genentech), Biclonic (Merus, WO2013157953); Dual Targeting domain antibodies (GSK/Domantis); Two-in-one Antibodies or Dual action Fabs recognizing two targets (Genentech, Novlmmune, Adimab); Cross-linked Mabs (Karmanos Cancer Center); covalently fused mAbs (AIMM), CovX-body (CovX/Pfizer); FynomAbs (Covagen/Janssen cilag); DutaMab (Dutalys/Roche); iMab (Medlmmune); IgG-like Bispecific (ImClone/Eli Lilly, Shen, J., et al. J Immunol Methods, 2007. 318(1-2) : p. 65-74); TIG-body, DIG-body and PIG-body

(Pharmabcine); Dual-affinity retargeting molecules (Fc-DART or Ig-DART, by Macrogenics, WO/2008/157379, WO/2010/080538); BEAT (Glenmark); Zybodies (Zyngenia); approaches with common light chain (Crucell/ Merus, US7262028) or common heavy chains (x Bodies by Novlmmune, W02012023053), as well as fusion proteins comprising a polypeptide sequence fused to an antibody fragment containing an Fc-domain like scFv-fusions, like BsAb by ZymoGenetics/BMS, HERCULES by Biogen Idee (US007951918), SCORPIONS by Emergent BioSolutions/Trubion and Zymogenetics/BMS, Ts2Ab (Medlmmune/AZ (Dimasi, N., et al. J Mol Biol, 2009. 393(3) : p. 672-92), scFv fusion by Genentech/Roche, scFv fusion by Novartis, scFv fusion by Immunomedics, scFv fusion by Changzhou Adam Biotech Inc (CN 102250246), TvAb by Roche (WO 2012025525, WO 2012025530), mAh ² by f-Star (W02008/003116), and dual scFv-fusions. It also should be understood that the term antibody, unless specified otherwise, also includes polyclonal antibodies, monoclonal antibodies (such as human monoclonal antibodies), antibody mixtures (recombinant polyclonals) for instance generated by technologies exploited by Symphogen and Merus (Oligoclonics), multimeric Fc proteins as described in WO2015/158867, fusion proteins as described in WO2014/031646 and antibody-like polypeptides, such as chimeric antibodies and humanized antibodies. An antibody as generated can potentially be of any isotype.

The terms "antigen-binding region", "antigen-binding site" or "antigen-binding domain", as used herein, refer to a region of a polypeptide, such as an antibody, which is capable of binding to an antigen . This binding region is typically defined by the VH and VL domains of an antibody which may be further subdivided into regions of hypervariability (or hypervariable regions which may be hypervariable in sequence and/or form of structurally defined loops), also termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs). The antigen can be any molecule, such as a polypeptide, e.g. present on a cell, bacterium, or virion.

The term "cell-associated antigen", when used herein, refers to an antigen which is associated to a cell rather than soluble in circulation . In one embodiment, the cell-associated antigen is a cell-surface-located antigen, e.g. an antigen exposed on the cell surface. In another embodiment, the cell-associated antigen is an integral membrane protein .

The term "full-length antibody" when used herein, refers to an antibody which contains all heavy and light chain constant and va riable domains corresponding to those that are normally found in a wild-type antibody of that isotype.

The term "human antibody", as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the invention may include a mino acid residues not encoded by human germline immunoglobulin sequences (e.g ., mutations, insertions or deletions introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo) . However, the term "human antibody", as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

The term "chimeric antibody", as used herein, refers to an antibody in which both chain types are chimeric as a result of antibody engineering. A chimeric chain is a chain that contains a foreign variable domain (originating from a non-human species, or synthetic or engineered from any species including human) linked to a constant region of human origin . The variable domain of a chimeric chain has a V region amino acid sequence which, analyzed as a whole, is closer to non-human species than to human .

The term "humanized antibody", as used herein, refers to an antibody in which both chain types are humanized as a result of antibody engineering. A humanized chain is typically a chain in which the complementarity determining regions (CDR) of the variable domains are foreign (originating from one species other than human, or synthetic) whereas the remainder of the cha in is of huma n origin. Humanization assessment is based on the resulting amino acid sequence, and not on the methodology per se, which allows protocols other than grafting to be used. The variable domain of a humanized chain has a V region amino acid sequence which, analyzed as a whole, is closer to human than to other species.

The terms "monoclonal antibody", "monoclonal Ab", "monoclonal antibody composition", "mAb", or the like, as used herein refer to a preparation of Ab molecules of single molecular composition . A monoclonal antibody composition displays a sing le binding specificity and affinity for a particular epitope. Accordingly, the term " human monoclonal antibody" refers to Abs displaying a sing le binding specificity which have variable and constant regions derived from human germline immunoglobulin sequences. The human mAbs may be generated by a hybridoma which includes a B cell obtained from a transgenic or trans-chromosoma I non-human animal, such as a transgenic mouse, having a genome comprising a human heavy chain transgene repertoire and a light chain transgene repertoire, rearranged to produce a functional human antibody and fused to an immortalized cell.

The term "isotype" as used herein, refers to the immunoglobulin class (for instance IgGl, IgG2, IgG3, IgG4, IgD, IgAl, IgGA2, IgE, or IgM or any allotypes thereof such as IgGlm(za) and IgGlm(f)) that is encoded by heavy chain constant region genes. Further, each heavy chain isotype can be combined with either a kappa (K) or lambda (l) light chain.

The term "mixed isotype" used herein refers to Fc region of an immunoglobulin generated by combining structural features of one isotype with the analogous region from another isotype thereby generating a hybrid isotype. A mixed isotype may comprise an Fc region having a sequence comprised of two or more isotypes selected from the following IgGl, IgG2, IgG3, IgG4, IgD, IgAl, IgGA2, IgE, or IgM thereby generating combinations such as e.g. IgGl/IgG3, IgGl/IgG4, IgG2/IgG3 or IgG2/IgG4.

The terms "antigen", "target antigen" or "antigen target" as used herein, refers to a molecule, such as a protein, to which the antigen-binding region of the polypeptide binds. An antigen molecule can contain one or more epitopes.

The term "epitope" means a protein determinant capable of specific binding to an antibody variable domain . Epitopes usually consist of surface groupings of molecules such as amino acids, sugar side chains or a combination thereof and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics. Conformational and non-conformational epitopes are distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents. The epitope may comprise amino acid residues directly involved in the binding (also called immunodominant component of the epitope) and other amino acid residues, which are not directly involved in the binding.

As used herein, the term "affinity" is the strength of binding of one molecule, e.g. an antibody, to another, e.g. a target or antigen, at a single site, such as the monovalent binding of an individual antigen binding site of an antibody to an antigen. As used herein, the term "avidity" refers to the combined strength of multiple binding sites between two structures, such as between multiple antigen-binding sites of antibodies simultaneously interacting with a target or e.g. between antibody and Clq. When more than one binding interactions are present, the two structures will only dissociate when all binding sites dissociate, and thus, the dissociation rate will be slower than for the individual binding sites, and thereby providing a greater effective total binding strength (avidity) compared to the strength of binding of the individual binding sites (affinity).

A "variant" or "polypeptide variant" or "antibody variant" in the present invention is a polypeptide or antibody molecule which comprises one or more mutations as compared to a reference antibody. Exemplary reference antibody formats include, without limitation, a wild-type antibody, such as a wild-type IgGl antibody, a full-length antibody or Fc-containing antibody fragment, a bispecific antibody, a human antibody, humanized antibody, chimeric antibody or any combination thereof. Exemplary mutations include amino acid deletions, insertions, and substitutions of amino acids in the parent amino acid sequence. Amino acid substitutions may exchange a native amino acid for another naturally-occurring amino acid, or for a non-naturally-occurring amino acid derivative. The amino acid substitution may be conservative or non-conservative. In the context of the present invention, conservative substitutions may be defined by substitutions within the classes of amino acids reflected in one or more of the following three tables:

Amino acid residue classes for conservative substitutions

Alternative conservative amino acid residue substitution classes

Alternative Physical and Functional Classifications of Amino Acid Residues

In the context of the present invention, a substitution in a variant is indicated as:

Original amino acid - position - substituted amino acid;

The three letter code, or one letter code, are used, including the codes Xaa and X to indicate amino acid residue. Accordingly, the notation "I253G" or "Ile253Gly" means that the variant comprises a substitution of Isoleucine with Glycine in the variant amino acid position corresponding to the amino acid in position 253 in the reference antibody.

Where a position as such is not present in an antibody, but the variant comprises an insertion of an amino acid, for example:

Position - inserted amino acid; the notation, e.g., "253G" is used.

Such notation is particularly relevant in connection with modification(s) in a series of homologous polypeptides or antibodies.

Similarly, when the identity of the substitution amino acid residue(s) is immaterial : Original amino acid - position; or "1253".

For a modification where the original amino acid(s) and/or substituted amino acid(s) may comprise more than one, but not all amino acid(s), the substitution of Isoleucine for Glycine, Lysine or Arginine in position 253: "Ile253Gly, Lys, Arg" or "I253G,K,R" or "I253G/K/R" or "1253 to G, K or R" may be used interchangeably in the context of the invention.

Furthermore, the term "a substitution" embraces a substitution into any one of the other nineteen natural amino acids, or into other amino acids, such as non- natural amino acids. For example, a substitution of amino acid E in position 345 includes each of the following substitutions: 345A, 345C, 345D, 345G, 345H, 345F, 3451, 345K, 345L, 345M, 345N, 345P, 345Q, 345R, 345S, 345T, 345V, 345W, and 345Y. This is equivalent to the designation 345X, wherein the X designates any amino acid. These substitutions can also be designated E345A, E345C, etc., or E345A,C,etc, or E345A/C/etc. The same applies to analogy to each and every position mentioned herein, to specifically include herein any one of such substitutions.

When used herein, the term "and/or" between options or embodiments is intended to cover all possible alternatives and combinations. E.g. "A and/or B and/or C" would be intended to cover all of the following embodiments:

A

B

C

A and B

A and C

B and C

A and B and C

As used herein, the term "effector cell" refers to an immune cell which is involved in the effector phase of an immune response, as opposed to the recognition and activation phases of an immune response. Exemplary immune cells include a cell of a myeloid or lymphoid origin, for instance lymphocytes (such as B cells and T cells including cytolytic T cells (CTLs)), killer cells, natural killer cells, macrophages, monocytes, eosinophils, polymorphonuclear cells, such as neutrophils, granulocytes, mast cells, and basophils. Some effector cells express Fc receptors (FcRs) or complement receptors and carry out specific immune functions. In some embodiments, an effector cell such as, e.g., a natural killer cell, is capable of inducing ADCC. For example, monocytes, macrophages, neutrophils, dendritic cells and Kupffer cells which express FcRs, are involved in specific killing of target cells and presenting antigens to other components of the immune system, or binding to cells that present antigens. In some embodiments, the ADCC can be further enhanced by antibody driven classical complement activation resulting in the deposition of activated C3 fragments on the target cell. C3 cleavage products are ligands to complement receptors (CRs), such as CR3, expressed on myeloid cells. The recognition of complement fragments by CRs on effector cells may promote enhanced Fc receptor-mediated ADCC. In some embodiments antibody driven classical complement activation leads to C3 fragments on the target cell. These C3 cleavage products may promote direct complement-dependent cellular cytotoxicity (CDCC). In some embodiments, an effector cell may phagocytose a target antigen, target particle or target cell. The expression of a particular FcR or complement receptor on an effector cell may be regulated by humoral factors such as cytokines. For example, expression of FcyRI has been found to be up-regulated by interferon y (IFN y) and/or G-CSF. This enhanced expression increases the cytotoxic activity of FcyRI-bearing cells against targets. An effector cell can phagocytose a target antigen or phagocytose or lyse a target cell. In some embodiments antibody driven classical complement activation leads to C3 fragments on the target cell. These C3 cleavage products may promote direct phagocytosis by effector cells or indirectly by enhancing antibody mediated phagocytosis.

The term "Fc-mediated effector functions," as used herein, is intended to refer to functions that are a consequence of binding a polypeptide or antibody to its target, such as an antigen, on a cell membrane wherein the Fc effector function is attributable to the Fc region of the polypeptide or antibody. Examples of Fc effector functions include (i) Clq-binding, (ii) complement activation, (iii) complement- dependent cytotoxicity (CDC), (iv) antibody-dependent cell-mediated cytotoxicity (ADCC), (v) Fc-gamma receptor-binding, (vi) antibody-dependent cellular phagocytosis (ADCP), (vii) complement-dependent cellular cytotoxicity (CDCC), (viii) complement-enhanced cytotoxicity, (ix) binding to complement receptor of a complement opsonized antibody mediated by the antibody, (x) opsonisation, and (xi) a combination of any of (i) to (x).

The term "vector," as used herein, refers to a nucleic acid molecule capable of inducing transcription of a nucleic acid segment ligated into the vector. One type of vector is a "plasmid", which is in the form of a circular double stranded DNA loop. Another type of vector is a viral vector, wherein the nucleic acid segment may be ligated into the viral genome.

The term "host cell" refers to a cell into which an expression vector has been introduced, as by transfection. It should be understood that such terms are intended to refer not only to the particular subject cell, but also to the progeny of such a cell. Host cells include, for example, CHO cells, HEK-293 cells, PER.C6, NSO cells, and lymphocytic cells, and prokaryotic cells such as E. coli and other eukaryotic hosts such as plant cells and fungi.

As used herein, the term "oligomer" refers to a molecule that consists of more than one but a limited number of monomer units (e.g. antibodies) in contrast to a polymer that, at least in principle, consists of an unlimited number of monomers. Exemplary oligomers are dimers, trimers, tetramers, pentamers and hexamers. Greek prefixes are often used to designate the number of monomer units in the oligomer, for example a tetramer being composed of four units and a hexamer of six units.

The term "oligomerization", as used herein, is intended to refer to a process that converts monomers to a finite degree of polymerization. Herein, it is observed, that, antibodies comprising target-binding regions according to the invention can form oligomers, such as hexamers, via non-covalent association of Fc-regions after target binding, e.g., at a cell surface. In the context of the present application, the terms "self-oligomerization", "auto-oligomerization" or "homo-oligomerization" may be used interchangeably and is intended to refer to a process of oligomerization between antibody molecules that have identical protein sequences disregarding post- translational modifications. The term "hetero-oligomerization", as used herein, is intended to refer to a process of oligomerization between antibody molecules that have different protein sequences disregarding post-translational modifications. Different antibodies participating in hetero-oligomerization could for instance bind different antigens, such as different target proteins, glycoproteins, glycans, or glycolipids.

The term "self-oligomerization inhibiting substitution" is intended to refer to a substitution in a polypeptide comprising an Fc region of an immunoglobulin and an antigen-binding region that inhibits the process of oligomerization between antibody molecules that have identical protein sequences disregarding post-translational modifications. Inhibition of self-oligomerization can be illustrated as an increase in EC50 of CDC activity or a reduction in maximal CDC lysis activity of the antibody, when measured according to the methods described in examples 5 and 9.

The term "clustering", as used herein, is intended to refer to oligomerization of antibodies, polypeptides, antigens or other proteins through non-covalent interactions.

The term "co-dependent", as used herein, is intended to refer to a functional effect that is dependent on the simultaneous binding of two or more different polypeptides with self-oligomerization inhibiting substitutions to a target on the same cell. In the context of the present invention, functional effects, such as CDC activity, can be dependent on the simultaneous binding of a first and second polypeptide i.e. the effect is said to be co-dependent. Thus, the effector function e.g. CDC activity of a first polypeptide having a self-oligomerization inhibiting substitution is dependent on the binding of a second polypeptide having a self-oligomerization inhibiting substitution, where the co-dependent effector function is present if said self- oligomerization substitutions are complementary.

When used herein, in the context of two antigens, the term "co-located" or grammatical variations thereof, is intended to refer, on one hand, to situations where the two antigens are co-expressed on the same cell. The antigens may already be adjacent to each other on the cell or the antigens may be brought together via oligomerization of the binding polypeptides, e.g. antibodies, of the invention. Furthermore, the term "co-located" is also intended to refer to situations wherein the two antigens are expressed on different cells, but wherein such cells are located in close proximity to each other.

As used herein, the term "complement activation" refers to the activation of the classical complement pathway, which is initiated by a large macromolecular complex called Cl binding to antibody-antigen complexes on a surface. Cl is a complex, which consists of the recognition protein Clq that is composed of 6 heterotrimeric subunits, and a hetero-tetramer of serine proteases, Clr2Cls2. Cl is the first protein complex in the early events of the classical complement cascade that involves a series of cleavage reactions that starts with the cleavage of C4 into C4a and C4b and C2 into C2a and C2b. C4b is deposited and forms together with C2a an enzymatic active convertase called C3 convertase, which cleaves complement component C3 into C3b and C3a, which forms a C5 convertase. This C5 convertase splits C5 in C5a and C5b and the last component is deposited on the membrane and that in turn triggers the late events of complement activation in which terminal complement components C5b, C6, C7, C8 and C9 assemble into the membrane attack complex (MAC). The complement cascade results in the creation of pores due to which causes cell lysis, also known as complement-dependent cytotoxicity (CDC). Complement activation can be evaluated by using Clq efficacy or CDC kinetics CDC assays (as described in W02013/004842, W02014/108198) or by the method Cellular deposition of C3b and C4b described in Beurskens et al April 1, 2012 vol. 188 no. 7 3532-3541.

The term "complement-dependent cytotoxicity" ("CDC"), as used herein, is intended to refer to the process of antibody-mediated complement activation leading to lysis of a cell or virion when antibody is bound to its target on the cell or virion as a result of pores in the membrane that are created by MAC assembly. CDC can be evaluated by in vitro assay such as a CDC assay in which normal human serum is used as a complement source with an antibody concentration series, as described in Example 2, 3, 4, 5 and 6 or in a Clq concentration series.

The term "antibody-dependent cell-mediated cytotoxicity" ("ADCC") as used herein, is intended to refer to a mechanism of killing of antibody-coated target cells or virions by cells expressing Fc receptors that recognize the Fc region of the bound antibody. ADCC can be determined using in vitro methods such as a chromium- release ADCC assay or a Luminescent ADCC Reporter BioAssay.

The term "antibody-drug conjugate", as used herein refers to an antibody or Fc-containing polypeptide having specificity for at least one type of malignant cell, a drug, and a linker coupling the drug to e.g. the antibody. The linker is cleavable or non-cleavable in the presence of the malignant cell; wherein the antibody-drug conjugate kills the malignant cell.

The term "antibody-drug conjugate uptake", as used herein refers to the process in which antibody-drug conjugates are bound to a target on a cell followed by uptake/engulfment by the cell membrane and thereby are drawn into the cell. Antibody-drug conjugate uptake may be evaluated as "antibody-mediated internalization and cell killing by anti-TF ADC in an in vitro killing assay" as described in WO 2011/157741.

The term "death receptor", as used herein refers to a member of the tumor necrosis factor receptor superfamily (TNFR-SF) comprising an intracellular death domain, including DR1, DR2 (also known as FAS), DR3, DR4, DR5, DR6, EDAR and NGFR. In humans, the DR1 protein is encoded by a nucleic acid sequence encoding the amino acid sequence UniprotKB/Swissprot P19438, the DR2 protein is encoded by a nucleic acid sequence encoding the amino acid sequence UniprotKB/Swissprot P25445, the DR3 protein is encoded by a nucleic acid sequence encoding the amino acid sequence UniprotKB/Swissprot Q93038, the DR4 protein is encoded by a nucleic acid sequence encoding the amino acid sequence UniprotKB/Swissprot 000220, the DR5 protein is encoded by a nucleic acid sequence encoding the amino acid sequence UniprotKB/Swissprot 014763), the DR6 protein is encoded by a nucleic acid sequence encoding the amino acid sequence UniprotKB/Swissprot 075509, the EDAR protein is encoded by a nucleic acid sequence encoding the amino acid sequence UniprotKB/Swissprot Q9UNE0, and the NGFR protein is encoded by a nucleic acid sequence encoding the amino acid sequence UniprotKB/Swissprot P08138. The death domains (DDs) are well-known protein interaction modules that belong to the death domain superfamily (Park Apoptosis. 2011 Mar; 16(3) : 209-20).

Further aspects and embodiments of the invention

As explained above, the invention is directed to a combination treatment involving two variant Fc-region-containing polypeptides, a first variant polypeptide and a second variant polypeptide, typically variant antibodies, which have been modified in their Fc regions so that hetero-oligomerization is favored over homo- oligomerization. That is, oligomerization between first variant molecules and second variant molecules is favored over oligomerization between first variant molecules and first variant molecules or oligomerization between second variant molecules and second variant molecules. This can be achieved by introducing modifications of positions corresponding to 253, 310, 436, 438, 439 and/or 440 in the Fc region of human IgGl, as described in further details herein .

Accordingly, the invention relates to a method of treating a disease or disorder comprising administering to a subject in need thereof: a first polypeptide comprising a first Fc region of a human IgG and a first antigen-binding region capable of binding to a first antigen, in combination with a second polypeptide comprising a second Fc region of a human IgG and a second antigen-binding region capable of binding to a second antigen, wherein

a) said first polypeptide comprises an I253G mutation of an amino acid position corresponding to 1253 in human IgGl and said second polypeptide comprises an H310R mutation of an amino acid position corresponding to H310 in human IgGl, or vice versa,

or said first polypeptide comprises an I253K or I253R mutation of an amino acid position corresponding to 1253 in human IgGl and said second polypeptide comprises an H310D mutation of an amino acid position corresponding to H310 in human IgGl, or vice versa,

and/or

b) said first polypeptide comprises a Y436N, Y436K, Y436Q or Y436R mutation of an amino acid position corresponding to Y436 in human IgGl and said second polypeptide comprises a Q438R, Q438K, Q438H, Q438G or Q438N mutation of an amino acid position corresponding to Q438 in human IgGl, or vice versa, or

said first polypeptide comprises a Y436N or Y436Q mutation of an amino acid position corresponding to Y436 in human IgGl and said second polypeptide comprises a Y436K or Y436R mutation of an amino acid position corresponding to Y436 in human IgGl, or vice versa,

or

said first polypeptide comprises a Q438R, Q438K or Q438H mutation of an amino acid position corresponding to Q438 in human IgGl and said second polypeptide comprises a Q438N or Q438G mutation of an amino acid position corresponding to Q438 in human IgGl, or vice versa,

and/or

c) said first polypeptide comprises a K439F, K439I, K439Y, K439T, K439V, K439W mutation of an amino acid position corresponding to K439 in human IgGl and said second polypeptide comprises an S440K mutation of an amino acid position corresponding to S440 in human IgGl, or vice versa,

wherein the amino acid positions correspond to human IgGl according to EU numbering.

In one aspect of the invention said first polypeptide comprises a F436N, F436K, F436Q or F436R mutation of an amino acid position corresponding to F436 in human IgG3 and said second polypeptide comprises a Q438R, Q438K, Q438H, Q438G or Q438N mutation of an amino acid position corresponding to Q438 in human IgGl, or vice versa,

or

said first polypeptide comprises a F436N or F436Q mutation of an amino acid position corresponding to F436 in human IgG3 and said second polypeptide comprises a F436K or F436R mutation of an amino acid position corresponding to F436 in human IgG3, or vice versa.

The amino acid in position 436 according to EU numbering is not conserved between IgGl and IgG3. Thus, amino acid position 436 in IgGl is a Tyrosine (Y) whereas the amino acid position 436 in IgG3 is a Phenylalanine (F).

In one embodiment of the method of invention, said first polypeptide comprises a Y436N, Y436K, Y436Q or Y436R mutation of an amino acid position corresponding to Y436 in human IgGl and said second polypeptide comprises a Q438R, Q438K, Q438H, Q438G or Q438N mutation of an amino acid position corresponding to Q438 in human IgGl, or vice versa,

or

said first polypeptide comprises a Y436N or Y436Q mutation of an amino acid position corresponding to Y436 in human IgGl and said second polypeptide comprises a Y436K or Y436R mutation of an amino acid position corresponding to Y436 in human IgGl, or vice versa,

or

said first polypeptide comprises a Q438R, Q438K or Q438H mutation of an amino acid position corresponding to Q438 in human IgGl and said second polypeptide comprises a Q438N or Q438G mutation of an amino acid position corresponding to Q438 in human IgGl, or vice versa,

wherein the amino acid positions correspond to human IgGl according to EU numbering.

In one embodiment of the method of invention, said first polypeptide comprises a Y436N, Y436K, Y436Q or Y436R mutation of an amino acid position corresponding to Y436 in human IgGl and said second polypeptide comprises a Q438R, Q438K, Q438H, Q438G or Q438N mutation of an amino acid position corresponding to Q438 in human IgGl, or vice versa, wherein the amino acid positions correspond to human IgGl according to EU numbering.

In one embodiment of the method of invention, said first polypeptide comprises a Y436N or Y436K, mutation of an amino acid position corresponding to Y436 in human IgGl and said second polypeptide comprises a Q438R or Q438N mutation of an amino acid position corresponding to Q438 in human IgGl, or vice versa,

or

said first polypeptide comprises a Q438R mutation of an amino acid position corresponding to Q438 in human IgGl and said second polypeptide comprises a Q438N mutation of an amino acid position corresponding to Q438 in human IgGl, or vice versa, wherein the amino acid positions correspond to human IgGl according to EU numbering.

As explained above, the Fc regions can be of a human IgGl, but also of a different human IgG, such as IgG2, IgG3 or IgG4. Figure 1 shows which positions in human IgG2, IgG3 and IgG4 correspond to which positions in IgGl.

An Fc region of a polypeptide used in the present invention is, like that of an antibody, comprised of two heavy chains. It is to be understood that when certain mutations in the Fc region are specified, said mutations are present in both chains of the Fc region.

In one embodiment of the method of the invention,

a) said first polypeptide comprises an I253G mutation of an amino acid position corresponding to 1253 in human IgGl and said second polypeptide comprises an H310R mutation of an amino acid position corresponding to H310 in human IgGl, or vice versa,

or

said first polypeptide comprises an I253K or I253R mutation of an amino acid position corresponding to 1253 in human IgGl and said second polypeptide comprises an H310D mutation of an amino acid position corresponding to H310 in human IgGl, or vice versa,

and/or

b) said first polypeptide comprises a Y436N mutation of an amino acid position corresponding to Y436 in human IgGl and said second polypeptide comprises a Q438R mutation of an amino acid position corresponding to Q438 in human IgGl, or vice versa,

or

said first polypeptide comprises a Y436N mutation of an amino acid position corresponding to Y436 in human IgGl and said second polypeptide comprises a Y436K mutation of an amino acid position corresponding to Y436 in human IgGl, or vice versa,

or

said first polypeptide comprises a Q438R mutation of an amino acid position corresponding to Q438 in human IgGl and said second polypeptide comprises a Q438N mutation of an amino acid position corresponding to Q438 in human IgGl, or vice versa, and/or

c) said first polypeptide comprises a K439F, K439I, K439Y, K439T, K439V, K439W mutation of an amino acid position corresponding to K439 in human IgGl and said second polypeptide comprises an S440K mutation of an amino acid position corresponding to S440 in human IgGl, or vice versa.

In one embodiment of the method of the invention, said first polypeptide comprises a Y436N or Y436K mutation of an amino acid position corresponding to Q436 in human IgGl and said second polypeptide comprises a Q438N or Q438R mutation of an amino acid position corresponding to Q438 in human IgGl, or vice versa.

In one embodiment of the method of the invention, said first polypeptide comprises a Y436N mutation of an amino acid position corresponding to Q436 in human IgGl and said second polypeptide comprises a Q438N or Q438R mutation of an amino acid position corresponding to Q438 in human IgGl, or vice versa.

In one embodiment of the method of the invention, said first polypeptide comprises a Y436N mutation of an amino acid position corresponding to Q436 in human IgGl and said second polypeptide comprises a Q438R mutation of an amino acid position corresponding to Q438 in human IgGl, or vice versa.

In one embodiment of the method of the invention, said first polypeptide comprises a Y436N mutation of an amino acid position corresponding to Q436 in human IgGl and said second polypeptide comprises a Q436K mutation of an amino acid position corresponding to Q436 in human IgGl, or vice versa.

In one embodiment of the method of the invention, said first polypeptide comprises a Y436K mutation of an amino acid position corresponding to Q436 in human IgGl and said second polypeptide comprises a Q438N or Q438R mutation of an amino acid position corresponding to Q438 in human IgGl, or vice versa.

In one embodiment of the method of the invention, said first polypeptide comprises a Y436K mutation of an amino acid position corresponding to Q436 in human IgGl and said second polypeptide comprises a Q438N mutation of an amino acid position corresponding to Q438 in human IgGl, or vice versa.

In one embodiment of the method of the invention, said first polypeptide comprises a Y436K mutation of an amino acid position corresponding to Q436 in human IgGl and said second polypeptide comprises a Q438R mutation of an amino acid position corresponding to Q438 in human IgGl, or vice versa. In one embodiment of the method of the invention, said first polypeptide comprises a Y438N mutation of an amino acid position corresponding to Q438 in human IgGl and said second polypeptide comprises a Q438R mutation of an amino acid position corresponding to Q438 in human IgGl, or vice versa.

In another embodiment of the method of the invention,

a) said first polypeptide comprises an I253G mutation of an amino acid position corresponding to 1253 in human IgGl and said second polypeptide comprises an H310R mutation of an amino acid position corresponding to H310 in human IgGl, or vice versa,

or

said first polypeptide comprises an I253K or I253R mutation of an amino acid position corresponding to 1253 in human IgGl and said second polypeptide comprises an H310D mutation of an amino acid position corresponding to H310 in human IgGl, or vice versa,

and

b) said first polypeptide comprises a Y436N, Y436K, Y436Q or Y436R mutation of an amino acid position corresponding to Y436 in human IgGl and said second polypeptide comprises a Q438R, Q438K, Q438H, Q438G or Q438N mutation of an amino acid position corresponding to Q438 in human IgGl, or vice versa, wherein preferably said first polypeptide comprises a Y436N mutation of an amino acid position corresponding to Y436 in human IgGl and said second polypeptide comprises a Q438R mutation of an amino acid position corresponding to Q438 in human IgGl, or vice versa,

or

said first polypeptide comprises a Y436N or Y436Q mutation of an amino acid position corresponding to Y436 in human IgGl and said second polypeptide comprises a Y436K or Y436R mutation of an amino acid position corresponding to Y436 in human IgGl, or vice versa, wherein preferably said first polypeptide comprises a Y436N mutation of an amino acid position corresponding to Y436 in human IgGl and said second polypeptide comprises a Y436K mutation of an amino acid position corresponding to Y436 in human IgGl, or vice versa, or

said first polypeptide comprises a Q438R, Q438K or Q438H mutation of an amino acid position corresponding to Q438 in human IgGl and said second polypeptide comprises a Q438N or Q438G mutation of an amino acid position corresponding to Q438 in human IgGl, or vice versa, wherein preferably said first polypeptide comprises a Q438R mutation of an amino acid position corresponding to Q438 in human IgGl and said second polypeptide comprises a Q438N mutation of an amino acid position corresponding to Q438 in human IgGl, or vice versa.

In another embodiment of the method of the invention,

a) said first polypeptide comprises an I253G mutation of an amino acid position corresponding to 1253 in human IgGl and said second polypeptide comprises an H310R mutation of an amino acid position corresponding to H310 in human IgGl, or vice versa,

or

said first polypeptide comprises an I253K or I253R mutation of an amino acid position corresponding to 1253 in human IgGl and said second polypeptide comprises an H310D mutation of an amino acid position corresponding to H310 in human IgGl, or vice versa,

and

c) said first polypeptide comprises a K439F, K439I, K439Y, K439T, K439V, K439W mutation of an amino acid position corresponding to K439 in human IgGl and said second polypeptide comprises an S440K mutation of an amino acid position corresponding to S440 in human IgGl, or vice versa.

In another embodiment of the method of the invention,

b) said first polypeptide comprises a Y436N, Y436K, Y436Q or Y436R mutation of an amino acid position corresponding to Y436 in human IgGl and said second polypeptide comprises a Q438R, Q438K, Q438H, Q438G or Q438N mutation of an amino acid position corresponding to Q438 in human IgGl, or vice versa, wherein preferably said first polypeptide comprises a Y436N mutation of an amino acid position corresponding to Y436 in human IgGl and said second polypeptide comprises a Q438R mutation of an amino acid position corresponding to Q438 in human IgGl, or vice versa,

or

said first polypeptide comprises a Y436N or Y436Q mutation of an amino acid position corresponding to Y436 in human IgGl and said second polypeptide comprises a Y436K or Y436R mutation of an amino acid position corresponding to Y436 in human IgGl, or vice versa, wherein preferably said first polypeptide comprises a Y436N mutation of an amino acid position corresponding to Y436 in human IgGl and said second polypeptide comprises a Y436K mutation of an amino acid position corresponding to Y436 in human IgGl, or vice versa, or

said first polypeptide comprises a Q438R, Q438K or Q438H mutation of an amino acid position corresponding to Q438 in human IgGl and said second polypeptide comprises a Q438N or Q438G mutation of an amino acid position corresponding to Q438 in human IgGl, or vice versa, wherein preferably said first polypeptide comprises a Q438R mutation of an amino acid position corresponding to Q438 in human IgGl and said second polypeptide comprises a Q438N mutation of an amino acid position corresponding to Q438 in human IgGl, or vice versa and

c) said first polypeptide comprises a K439F, K439I, K439Y, K439T, K439V, K439W mutation of an amino acid position corresponding to K439 in human IgGl and said second polypeptide comprises an S440K mutation of an amino acid position corresponding to S440 in human IgGl, or vice versa.

In another embodiment of the method of the invention,

a) said first polypeptide comprises an I253G mutation of an amino acid position corresponding to 1253 in human IgGl and said second polypeptide comprises an H310R mutation of an amino acid position corresponding to H310 in human IgGl, or vice versa,

or

said first polypeptide comprises an I253K or I253R mutation of an amino acid position corresponding to 1253 in human IgGl and said second polypeptide comprises an H310D mutation of an amino acid position corresponding to H310 in human IgGl, or vice versa,

and

b) said first polypeptide comprises a Y436N, Y436K, Y436Q or Y436R mutation of an amino acid position corresponding to Y436 in human IgGl and said second polypeptide comprises a Q438R, Q438K, Q438H, Q438G or Q438N mutation of an amino acid position corresponding to Q438 in human IgGl, or vice versa, wherein preferably said first polypeptide comprises a Y436N mutation of an amino acid position corresponding to Y436 in human IgGl and said second polypeptide comprises a Q438R mutation of an amino acid position corresponding to Q438 in human IgGl, or vice versa,

or said first polypeptide comprises a Y436N or Y436Q mutation of an amino acid position corresponding to Y436 in human IgGl and said second polypeptide comprises a Y436K or Y436R mutation of an amino acid position corresponding to Y436 in human IgGl, or vice versa, wherein preferably said first polypeptide comprises a Y436N mutation of an amino acid position corresponding to Y436 in human IgGl and said second polypeptide comprises a Y436K mutation of an amino acid position corresponding to Y436 in human IgGl, or vice versa, or

said first polypeptide comprises a Q438R, Q438K or Q438H mutation of an amino acid position corresponding to Q438 in human IgGl and said second polypeptide comprises a Q438N or Q438G mutation of an amino acid position corresponding to Q438 in human IgGl, or vice versa, wherein preferably said first polypeptide comprises a Q438R mutation of an amino acid position corresponding to Q438 in human IgGl and said second polypeptide comprises a Q438N mutation of an amino acid position corresponding to Q438 in human IgGl, or vice versa, and

c) said first polypeptide comprises a K439F, K439I, K439Y, K439T, K439V, K439W mutation of an amino acid position corresponding to K439 in human IgGl and said second polypeptide comprises an S440K mutation of an amino acid position corresponding to S440 in human IgGl, or vice versa.

In a further embodiment of the method of the invention, the first and second polypeptides do not comprise the mutations specified in option c), but said first polypeptide further comprises a K439E mutation of an amino acid position corresponding to K439 in human IgGl and said second polypeptide further comprises an S440K mutation of an amino acid position corresponding to S440 in human IgGl, or vice versa.

In another embodiments of the method of the invention,

i) said first polypeptide comprises an I253G mutation of an amino acid position corresponding to 1253 in human IgGl and said second polypeptide comprises an H310R mutation of an amino acid position corresponding to H310 in human IgGl, or vice versa,

or

ii) said first polypeptide comprises an I253R mutation of an amino acid position corresponding to 1253 in human IgGl and said second polypeptide comprises an H310D mutation of an amino acid position corresponding to H310 in human IgGl, or vice versa,

or

iii) said first polypeptide comprises an I253G mutation of an amino acid position corresponding to 1253 in human IgGl and said second polypeptide comprises an H310R mutation of an amino acid position corresponding to H310 in human IgGl, or vice versa, and said first polypeptide comprises a K439E mutation of an amino acid position corresponding to K439 in human IgGl and said second polypeptide comprises an S440K mutation of an amino acid position corresponding to S440 in human IgGl, or vice versa,

or

iv) said first polypeptide comprises an I253R mutation of an amino acid position corresponding to 1253 in human IgGl and said second polypeptide comprises an H310D mutation of an amino acid position corresponding to H310 in human IgGl, or vice versa, and said first polypeptide comprises a K439E mutation of an amino acid position corresponding to K439 in human IgGl and said second polypeptide comprises an S440K mutation of an amino acid position corresponding to S440 in human IgGl, or vice versa,

or

v) said first polypeptide comprises a Y436N mutation of an amino acid position corresponding to Y436 in human IgGl and said second polypeptide comprises a Q438R mutation of an amino acid position corresponding to Q438 in human IgGl, or vice versa,

or

vi) said first polypeptide comprises a Y436N mutation of an amino acid position corresponding to Y436 in human IgGl and said second polypeptide comprises a Y436K mutation of an amino acid position corresponding to Y436 in human IgGl, or vice versa,

or

vii) said first polypeptide comprises a Q438R mutation of an amino acid position corresponding to Q438 in human IgGl and said second polypeptide comprises a Q438N mutation of an amino acid position corresponding to Q438 in human IgGl, or vice versa,

or viii) said first polypeptide comprises a Y436N mutation of an amino acid position corresponding to Y436 in human IgGl and said second polypeptide comprises a Q438R mutation of an amino acid position corresponding to Q438 in human IgGl, or vice versa, and said first polypeptide comprises an I253G mutation of an amino acid position corresponding to 1253 in human IgGl and said second polypeptide comprises an H310R mutation of an amino acid position corresponding to H310 in human IgGl, or vice versa,

or

ix) said first polypeptide comprises a Y436N mutation of an amino acid position corresponding to Y436 in human IgGl and said second polypeptide comprises a Q438R mutation of an amino acid position corresponding to Q438 in human IgGl, or vice versa, said first polypeptide comprises an I253R mutation of an amino acid position corresponding to 1253 in human IgGl and said second polypeptide comprises an H310D mutation of an amino acid position corresponding to H310 in human IgGl, or vice versa,

or

x) said first polypeptide comprises a Y436N mutation of an amino acid position corresponding to Y436 in human IgGl and said second polypeptide comprises a Q438R mutation of an amino acid position corresponding to Q438 in human IgGl, or vice versa, and said first polypeptide comprises an I253G mutation of an amino acid position corresponding to 1253 in human IgGl and said second polypeptide comprises an H310R mutation of an amino acid position corresponding to H310 in human IgGl, or vice versa, and said first polypeptide comprises a K439E mutation of an amino acid position corresponding to K439 in human IgGl and said second polypeptide comprises an S440K mutation of an amino acid position corresponding to S440 in human IgGl, or vice versa,

or

xi) said first polypeptide comprises a Y436N mutation of an amino acid position corresponding to Y436 in human IgGl and said second polypeptide comprises a Q438R mutation of an amino acid position corresponding to Q438 in human IgGl, or vice versa, and said first polypeptide comprises an I253R mutation of an amino acid position corresponding to 1253 in human IgGl and said second polypeptide comprises an H310D mutation of an amino acid position corresponding to H310 in human IgGl, or vice versa, and said first polypeptide comprises a K439E mutation of an amino acid position corresponding to K439 in human IgGl and said second polypeptide comprises an S440K mutation of an amino acid position corresponding to S440 in human IgGl, or vice versa,

or

xii) said first polypeptide comprises a Y436N mutation of an amino acid position corresponding to Y436 in human IgGl and said second polypeptide comprises a Y436K mutation of an amino acid position corresponding to Y436 in human IgGl, or vice versa, said first polypeptide comprises an I253G mutation of an amino acid position corresponding to 1253 in human IgGl and said second polypeptide comprises an H310R mutation of an amino acid position corresponding to H310 in human IgGl, or vice versa,

or

xiii) said first polypeptide comprises a Y436N mutation of an amino acid position corresponding to Y436 in human IgGl and said second polypeptide comprises a Y436K mutation of an amino acid position corresponding to Y436 in human IgGl, or vice versa, said first polypeptide comprises an I253R mutation of an amino acid position corresponding to 1253 in human IgGl and said second polypeptide comprises an H310D mutation of an amino acid position corresponding to H310 in human IgGl, or vice versa,

or

xiv) said first polypeptide comprises a Y436N mutation of an amino acid position corresponding to Y436 in human IgGl and said second polypeptide comprises a Y436K mutation of an amino acid position corresponding to Y436 in human IgGl, or vice versa, said first polypeptide comprises an I253G mutation of an amino acid position corresponding to 1253 in human IgGl and said second polypeptide comprises an H310R mutation of an amino acid position corresponding to H310 in human IgGl, or vice versa, and said first polypeptide comprises a K439E mutation of an amino acid position corresponding to K439 in human IgGl and said second polypeptide comprises an S440K mutation of an amino acid position corresponding to S440 in human IgGl, or vice versa,

or

xv) said first polypeptide comprises a Y436N mutation of an amino acid position corresponding to Y436 in human IgGl and said second polypeptide comprises a Y436K mutation of an amino acid position corresponding to Y436 in human IgGl, or vice versa, said first polypeptide comprises an I253R mutation of an amino acid position corresponding to 1253 in human IgGl and said second polypeptide comprises an H310D mutation of an amino acid position corresponding to H310 in human IgGl, or vice versa, and said first polypeptide comprises a K439E mutation of an amino acid position corresponding to K439 in human IgGl and said second polypeptide comprises an S440K mutation of an amino acid position corresponding to S440 in human IgGl, or vice versa,

or

xvi) said first polypeptide comprises a Q438R mutation of an amino acid position corresponding to Q438 in human IgGl and said second polypeptide comprises a Q438N mutation of an amino acid position corresponding to Q438 in human IgGl, or vice versa, said first polypeptide comprises an I253G mutation of an amino acid position corresponding to 1253 in human IgGl and said second polypeptide comprises an H310R mutation of an amino acid position corresponding to H310 in human IgGl, or vice versa,

or

xvii) said first polypeptide comprises a Q438R mutation of an amino acid position corresponding to Q438 in human IgGl and said second polypeptide comprises a Q438N mutation of an amino acid position corresponding to Q438 in human IgGl, or vice versa, said first polypeptide comprises an I253R mutation of an amino acid position corresponding to 1253 in human IgGl and said second polypeptide comprises an H310D mutation of an amino acid position corresponding to H310 in human IgGl, or vice versa,

or

xviii) said first polypeptide comprises a Q438R mutation of an amino acid position corresponding to Q438 in human IgGl and said second polypeptide comprises a Q438N mutation of an amino acid position corresponding to Q438 in human IgGl, or vice versa, said first polypeptide comprises an I253G mutation of an amino acid position corresponding to 1253 in human IgGl and said second polypeptide comprises an H310R mutation of an amino acid position corresponding to H310 in human IgGl, or vice versa, and said first polypeptide comprises a K439E mutation of an amino acid position corresponding to K439 in human IgGl and said second polypeptide comprises an S440K mutation of an amino acid position corresponding to S440 in human IgGl, or vice versa,

or

xix) said first polypeptide comprises a Q438R mutation of an amino acid position corresponding to Q438 in human IgGl and said second polypeptide comprises a Q438N mutation of an amino acid position corresponding to Q438 in human IgGl, or vice versa, said first polypeptide comprises an I253R mutation of an amino acid position corresponding to 1253 in human IgGl and said second polypeptide comprises an H310D mutation of an amino acid position corresponding to H310 in human IgGl, or vice versa, and said first polypeptide comprises a K439E mutation of an amino acid position corresponding to K439 in human IgGl and said second polypeptide comprises an S440K mutation of an amino acid position corresponding to S440 in human IgGl, or vice versa,

or

xx) said first polypeptide comprises a Y436N mutation of an amino acid position corresponding to Y436 in human IgGl and said second polypeptide comprises a Q438R mutation of an amino acid position corresponding to Q438 in human IgGl, or vice versa, and said first polypeptide comprises a K439E mutation of an amino acid position corresponding to K439 in human IgGl and said second polypeptide comprises an S440K mutation of an amino acid position corresponding to S440 in human IgGl, or vice versa,

or

xxi) said first polypeptide comprises a Y436N mutation of an amino acid position corresponding to Y436 in human IgGl and said second polypeptide comprises a Y436K mutation of an amino acid position corresponding to Y436 in human IgGl, or vice versa, and said first polypeptide comprises a K439E mutation of an amino acid position corresponding to K439 in human IgGl and said second polypeptide comprises an S440K mutation of an amino acid position corresponding to S440 in human IgGl, or vice versa,

or

xxii) said first polypeptide comprises a Q438R mutation of an amino acid position corresponding to Q438 in human IgGl and said second polypeptide comprises a Q438N mutation of an amino acid position corresponding to Q438 in human IgGl, or vice versa, and said first polypeptide comprises a K439E mutation of an amino acid position corresponding to K439 in human IgGl and said second polypeptide comprises an S440K mutation of an amino acid position corresponding to S440 in human IgGl, or vice versa.

In preferred embodiments of the method of the invention,

i) said first polypeptide comprises a Y436N mutation of an amino acid position corresponding to Y436 in human IgGl and said second polypeptide comprises a Q438R mutation of an amino acid position corresponding to Q438 in human IgGl, or vice versa,

or

ii) said first polypeptide comprises a Y436N mutation of an amino acid position corresponding to Y436 in human IgGl and said second polypeptide comprises a Q438N mutation of an amino acid position corresponding to Q438 in human IgGl, or vice versa,

or

iii) said first polypeptide comprises a Y436N mutation of an amino acid position corresponding to Y436 in human IgGl and said second polypeptide comprises a Y436K mutation of an amino acid position corresponding to Y436 in human IgGl, or vice versa,

or

iv) said first polypeptide comprises a Y436K mutation of an amino acid position corresponding to Y436 in human IgGl and said second polypeptide comprises a Q438R mutation of an amino acid position corresponding to Q438 in human IgGl, or vice versa,

or

v) said first polypeptide comprises a Y436K mutation of an amino acid position corresponding to Y436 in human IgGl and said second polypeptide comprises a Q438N mutation of an amino acid position corresponding to Q438 in human IgGl, or vice versa,

or

vi) said first polypeptide comprises a Q438R mutation of an amino acid position corresponding to Q438 in human IgGl and said second polypeptide comprises a Q438N mutation of an amino acid position corresponding to Q438 in human IgGl, or vice versa,

and

vii) said first polypeptide comprises a K439E mutation of an amino acid position corresponding to K439 in human IgGl and said second polypeptide comprises an S440K mutation of an amino acid position corresponding to S440 in human IgGl, or vice versa.

In one embodiment of the method of the invention,

i) said first polypeptide comprises a Y436N or Y436K mutation of an amino acid position corresponding to Q436 in human IgGl and said second polypeptide comprises a Q438N or Q438R mutation of an amino acid position corresponding to

Q438 in human IgGl, or vice versa,

and

ii) said first polypeptide comprises a K439E mutation of an amino acid position corresponding to K439 in human IgGl and said second polypeptide comprises an S440K mutation of an amino acid position corresponding to S440 in human IgGl, or vice versa.

In one embodiment of the method of the invention,

i) said first polypeptide comprises a Y436N mutation of an amino acid position corresponding to Q436 in human IgGl and said second polypeptide comprises a Q438R mutation of an amino acid position corresponding to Q438 in human IgGl, or vice versa, and

ii) said first polypeptide comprises a K439E mutation of an amino acid position corresponding to K439 in human IgGl and said second polypeptide comprises an S440K mutation of an amino acid position corresponding to S440 in human IgGl, or vice versa.

In one embodiment of the method of the invention,

i) said first polypeptide comprises a Y438N mutation of an amino acid position corresponding to Q438 in human IgGl and said second polypeptide comprises a Q438R mutation of an amino acid position corresponding to Q438 in human IgGl, or vice versa, and

ii) said first polypeptide comprises a K439E mutation of an amino acid position corresponding to K439 in human IgGl and said second polypeptide comprises an S440K mutation of an amino acid position corresponding to S440 in human IgGl, or vice versa.

The following Table provides a non-limiting list of embodiments of the method of the invention, describing combinations of a first polypeptide and a second polypeptide with specific mutations, Thus, for example, embodiment 1 of the Table below is a combination of a first polypeptide comprising I253G and K439E mutations at positions corresponding to 1253 and K439, respectively, in human IgGl, with a second polypeptide comprising H310R and S440K mutations at positions corresponding to H310 and S440, respectively, in human IgGl. As described herein, the first and second polypeptides of all of the embodiments 1 to 288 can optionally comprise further mutations, such as oligomerization-enhancing mutations, e.g. E430G.

Optional further modifications

In some embodiments, one or both polypeptides used in the invention comprise further mutations that enhance oligomerization, such as hexamerization. Such mutations have e.g . been described in W02013/004842 and W02014/108198. By including such further mutations, the propensity of the first and second polypeptides to form hetero-oligomers, such as hetero-hexamers, will be even further enhanced. Examples of amino acid mutations that enhance Fc-Fc interaction between polypeptides and thereby oligomer formation are E430G, E345K, E430S, E430F, E430T, E345Q, E345R, E345Y, S440W, S440Y, T437R and K248E. Thus, such mutations promote or enhance oligomer formation, such as hexamer formation, and may also be described as Fc-Fc interaction enhancing mutations, hexmerization enhancing mutations or self-oligomerization enhancing mutations.

Thus, in some embodiments, said first polypeptide further comprises a mutation of an amino acid position corresponding to E430, E345, S440, T437 or K248 in human IgGl, and/or said second polypeptide further comprises a mutation of an amino acid position corresponding to E430, E345, S440, T437 or K248 in human IgGl, or vice versa, with the proviso that if said first or second polypeptide comprises a K439E, K439D, S440K, S440R or S440H mutation, said further mutation in said polypeptide is not at position S440.

In some of these embodiments, said further mutation in said first polypeptide is selected from the group consisting of: E430G, E345K, E430S, E430F, E430T, E345Q, E345R, E345Y, S440W and S440Y.

In some of these embodiments, said further mutation in said second polypeptide is selected from the group consisting of: E430G, E345K, E430S, E430F, E430T, E345Q, E345R, E345Y, S440W and S440Y.

In some of these embodiments, said further mutation in said first polypeptide is selected from the group consisting of: E430G, E345K, E430S, E430F, E430T, E345Q, E345R, E345Y, S440W and S440Y, and said further mutation in said second polypeptide is selected from the group consisting of: E430G, E345K, E430S, E430F, E430T, E345Q, E345R, E345Y, S440W and S440Y,

and/or

said first polypeptide comprises a T437R and a K248E mutation, and/or said second polypeptide comprises a T437R and a K248E mutation .

In some embodiments, said further mutation in said first polypeptide is selected from the group consisting of: E430G, E345K and E345R, and said further mutation in said second polypeptide is selected from the group consisting of: E430G, E345K and E345K. The further mutation may be independently selected from the group for said first and second polypeptide.

In some embodiments, said further mutation in said first polypeptide is selected from the group consisting of: E430G and E345K, and said further mutation in said second polypeptide is selected from the group consisting of: E430G and E345K. In a preferred embodiment, said further mutation in said first polypeptide is E430G and said further mutation in said second polypeptide E430G. In one embodiment, said further mutation in said first polypeptide is E345K and said further mutation in said second polypeptide E345K. In one embodiment, said further mutation in said first polypeptide is E345R and said further mutation in said second polypeptide E345R.

In one embodiment, said first polypeptide comprises a T437R and a K248E mutation, and said second polypeptide comprises a T437R and a K248E mutation. In one embodiment of the invention, said first polypeptide comprises an E430G mutation and said second polypeptide comprises an E430G mutation.

In another embodiment of the invention,

i) said first polypeptide comprises a Y436N mutation of an amino acid position corresponding to Y436 in human IgGl and said second polypeptide comprises a Q438R mutation of an amino acid position corresponding to Q438 in human IgGl, or vice versa,

or

ii) said first polypeptide comprises a Y436N mutation of an amino acid position corresponding to Y436 in human IgGl and said second polypeptide comprises a Q438N mutation of an amino acid position corresponding to Q438 in human IgGl, or vice versa,

or

iii) said first polypeptide comprises a Y436N mutation of an amino acid position corresponding to Y436 in human IgGl and said second polypeptide comprises a Y436K mutation of an amino acid position corresponding to Y436 in human IgGl, or vice versa,

or

iv) said first polypeptide comprises a Y436K mutation of an amino acid position corresponding to Y436 in human IgGl and said second polypeptide comprises a Q438R mutation of an amino acid position corresponding to Q438 in human IgGl, or vice versa,

or

v) said first polypeptide comprises a Y436K mutation of an amino acid position corresponding to Y436 in human IgGl and said second polypeptide comprises a Q438N mutation of an amino acid position corresponding to Q438 in human IgGl, or vice versa,

or

vi) said first polypeptide comprises a Q438R mutation of an amino acid position corresponding to Q438 in human IgGl and said second polypeptide comprises a Q438N mutation of an amino acid position corresponding to Q438 in human IgGl, or vice versa,

and

vii) said first polypeptide comprises a K439E mutation of an amino acid position corresponding to K439 in human IgGl and said second polypeptide comprises an S440K mutation of an amino acid position corresponding to S440 in human IgGl, or vice versa,

and

viii) said first polypeptide comprises a E430G, E345K or E345R mutation of an amino acid position corresponding to K430 or E345 in human IgGl and said second polypeptide comprises an E430G, E345K or E345R mutation of an amino acid position corresponding to K430 or E345 in human IgGl, or vice versa.

The E430G, E345K or E345R mutations may be independently selected for said first and second polypeptide. Thus, said first and second polypeptide may have the same mutation or a different mutation selected from the group consisting of: E430G, E345K or E345R.

In another embodiment of the invention,

i) said first polypeptide comprises a Y436N or Y436K mutation of an amino acid position corresponding to Q436 in human IgGl and said second polypeptide comprises a Q438N or Q438R mutation of an amino acid position corresponding to Q438 in human IgGl, or vice versa,

and

ii) said first polypeptide comprises a K439E mutation of an amino acid position corresponding to K439 in human IgGl and said second polypeptide comprises an S440K mutation of an amino acid position corresponding to S440 in human IgGl, or vice versa

and

iii) said first polypeptide comprises a E430G or E345K mutation of an amino acid position corresponding to K430 or E345 in human IgGl and said second polypeptide comprises an E430G or E345R mutation of an amino acid position corresponding to K430 or E345 in human IgGl, or vice versa.

In another embodiment of the invention,

i) said first polypeptide comprises a Y436N or Y436K mutation of an amino acid position corresponding to Q436 in human IgGl and said second polypeptide comprises a Q438N or Q438R mutation of an amino acid position corresponding to Q438 in human IgGl, or vice versa,

and

ii) said first polypeptide comprises a K439E mutation of an amino acid position corresponding to K439 in human IgGl and said second polypeptide comprises an S440K mutation of an amino acid position corresponding to S440 in human IgGl, or vice versa

and

iii) said first and second polypeptide comprises a E430G mutation of an amino acid position corresponding to K430 in human IgGl.

In another embodiment of the invention, said first polypeptide comprises a Y436N, K439E and E430G mutation wherein the amino acid positions correspond to Y436, K439 and E430 respectively in human IgGl and said second polypeptide comprises a Q438R, S440K and E430G mutation wherein the amino acid positions correspond to Q438, S440 and E430 in human IgGl, or vice versa.

In another embodiment of the invention, said first polypeptide comprises a Y436K, K439E and E430G mutation wherein the amino acid positions correspond to Y436, K439 and E430 respectively in human IgGl and said second polypeptide comprises a Q438N, S440K and E430G mutation wherein the amino acid positions correspond to Q438, S440 and E430 in human IgGl, or vice versa.

In another embodiment of the invention, said first polypeptide comprises a Y436K, K439E and E430G mutation wherein the amino acid positions correspond to Y436, K439 and E430 respectively in human IgGl and said second polypeptide comprises a Q438R, S440K and E430G mutation wherein the amino acid positions correspond to Q438, S440 and E430 in human IgGl, or vice versa.

In some embodiments, one or both polypeptides used in the invention comprise(s) further mutations that alter the ability of the polypeptide induce or mediate effector functions, such as Fc-mediated effector functions, e.g. CDC or ADCC. Such an altered ability can be an increased ability to induce effector functions or a decreased ability to induce effector functions. In some embodiments, one or both polypeptides used in the invention comprise further mutations that alter the ability of the polypeptide to bind Fc gamma receptors. Mutations that alter the ability of an antibody to induce effector functions and/or bind Fc gamma recptors have been described in the art. By including such further mutations, the propensity of the first and second variant polypeptides to induce effector functions will be increased or decreased and thus may be modulated according to what is desired in the given situation. For example, it can be desirable to introduce mutations that increase the ability of the polypeptides to induce CDC to even further promote the efficacy of hetero-oligomers. In some other situations, it may e.g. be a priority to further reduce toxicity of homo-oligomers by introducing mutations that reduce the ability to induce CDC.

Accordingly, in some embodiments of the invention, said first polypeptide and/or said second polypeptide has been further modified so that the polypeptide has an altered ability to induce effector functions, such as Fc-mediated effector functions, compared to a polypeptide which is identical except for said further modification.

In some embodiments, said first polypeptide and/or said second polypeptide has been further modified so that the polypeptide has an altered ability to induce antibody-dependent cell-mediated cytotoxicity compared to a polypeptide which is identical except for said further modification.

In other embodiments, said first polypeptide and/or said second polypeptide has been further modified so that the polypeptide has an altered ability to induce complement-dependent cytotoxicity compared to a polypeptide which is identical except for said modification.

Polypeptide formats

As described above, in a preferred embodiment of the method of the invention, said first polypeptide is an antibody. In another preferred embodiment of the invention, said second polypeptide is an antibody. In a more preferred embodiment, said first polypeptide is an antibody and said second polypeptide is an antibody.

In a further embodiment, said first polypeptide is a full-length antibody and/or said second polypeptide is a full-length antibody.

The Fc region or antibody may be of any IgG isotype, e.g. IgGl, IgG2, IgG3 or IgG4. In one embodiment of the invention the polypeptide or antibody has an Fc region that is a human IgGl, IgG2, IgG3 or IgG4 isotype. In one embodiment of the invention the Fc region is a mixed isotype, such as a mixed isotype selected from the group consisting of: IgGl/IgG2, IgGl/IgG3, IgGl/IgG4, IgG2/IgG3, IgG2/IgG4 and IgG3/IgG4. In a mixed isotype, the Fc region is comprised of an amino acid sequence from more than one isotype.

In preferred embodiments, said first polypeptide is an IgGl antibody and/or said second polypeptide is an IgGl antibody.

In one embodiment of the invention, the first and/or second polypeptide comprises a first and/or second Fc region comprising the sequence as set forth in SEQ ID NO: 22, 23, 24, 25, 31, 32, and 33, wherein at least one mutation according to the invention has been introduced into said sequence. The first and second Fc region may be independently selected from the sequences as set forth in SEQ ID NO:

22, 23, 24, 25, 31, 32, and 33. Thus, the first and second Fc region may be of the same parent sequence or of a different parent sequence.

In one embodiment of the invention, the first and/or second polypeptide comprises a first and/or second Fc region comprising the sequence as set forth in SEQ ID NO: 22, 23, 24 and 25, wherein at least one mutation according to the invention has been introduced into said sequence.

In one embodiment of the invention, the first polypeptide comprises a first Fc region comprising a sequence selected from the group consisting of SEQ ID NO: 22,

23, 24, 25, 26, 27, 28, 29, 30, 31, 32, and 33, wherein at least one mutation according to the invention has been introduced. In one embodiment of the invention the second polypeptide comprises a second Fc region comprising a sequence selected from the group consisting of SEQ ID NO: 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, and 33, wherein at least one mutation according to the invention has been introduced.

In one embodiment of the invention, the first polypeptide comprises a first Fc region comprising a sequence selected from the group consisting of SEQ ID NO: 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, and 33, wherein at least two mutations, or at least three mutations, according to the invention has been introduced. In one embodiment of the invention the second polypeptide comprises a second Fc region comprising a sequence selected from the group consisting of SEQ ID NO: 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, and 33 wherein at least two mutations, or at least three mutations, according to the invention has been introduced.

In one embodiment of the invention, the first polypeptide comprises a first Fc region comprising a sequence selected from the group consisting of: SEQ ID NO 63 ,64, 65, 66, 67, 68, 69, 70, 71, 72, 74, 75, 76, 77, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110. In one embodiment of the invention the second polypeptide comprises a second Fc region comprising a sequence selected from the group consisting of: SEQ ID NO 63 ,64, 65, 66, 67, 68, 69, 70, 71, 72, 74, 75, 76, 77, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110.

In one embodiment of the invention the i) first polypeptide comprises a first Fc region comprising a sequence selected from the group consisting of: 74, 76, 79 and 81, and

ii) the second polypeptide comprises a second Fc region comprising a sequence selected from the group consisting of: 75, 77, 80 and 82, or vice versa,

wherein the first and second Fc region has at most 5 further mutation (s), such as at most 4, such as at most 3 such as at most 2 such as at most one.

In one embodiment of the invention, the second polypeptide comprises a second Fc region comprising a sequence as set forth in SEQ ID NO : 63. In one embodiment of the invention the second polypeptide comprises a first Fc region comprising a sequence as set forth in SEQ ID NO: 64. In one embodiment of the invention the first polypeptide comprises a first Fc region comprising a sequence as set forth in SEQ ID NO: 65. In one embodiment of the invention the first polypeptide comprises a first Fc region comprising a sequence as set forth in SEQ ID NO: 66. In one embodiment of the invention the first polypeptide comprises a first Fc region comprising a sequence as set forth in SEQ ID NO: 67. In one embodiment of the invention the first polypeptide comprises a first Fc region comprising a sequence as set forth in SEQ ID NO: 68. In one embodiment of the invention the first polypeptide comprises a first Fc region comprising a sequence as set forth in SEQ ID NO: 69. In one embodiment of the invention the first polypeptide comprises a first Fc region comprising a sequence as set forth in SEQ ID NO: 70. In one embodiment of the invention the first polypeptide comprises a first Fc region comprising a sequence as set forth in SEQ ID NO: 71. In one embodiment of the invention the first polypeptide comprises a first Fc region comprising a sequence as set forth in SEQ ID NO: 72. In one embodiment of the invention the first polypeptide comprises a first Fc region comprising a sequence as set forth in SEQ ID NO: 74. In one embodiment of the invention the first polypeptide comprises a first Fc region comprising a sequence as set forth in SEQ ID NO: 75. In one embodiment of the invention the first polypeptide comprises a first Fc region comprising a sequence as set forth in SEQ ID NO: 76. In one embodiment of the invention the first polypeptide comprises a first Fc region comprising a sequence as set forth in SEQ ID NO: 77. In one embodiment of the invention the first polypeptide comprises a first Fc region comprising a sequence as set forth in SEQ ID NO: 79. In one embodiment of the invention the first polypeptide comprises a first Fc region comprising a sequence as set forth in SEQ ID NO: 80. In one embodiment of the invention the first polypeptide comprises a first Fc region comprising a sequence as set forth in SEQ ID NO: 81. In one embodiment of the invention the first polypeptide comprises a first Fc region comprising a sequence as set forth in SEQ ID NO: 82. In one embodiment of the invention the first polypeptide comprises a first Fc region comprising a sequence as set forth in SEQ ID NO: 83. In one embodiment of the invention the first polypeptide comprises a first Fc region comprising a sequence as set forth in SEQ ID NO: 84. In one embodiment of the invention the first polypeptide comprises a first Fc region comprising a sequence as set forth in SEQ ID NO: 85. In one embodiment of the invention the first polypeptide comprises a first Fc region comprising a sequence as set forth in SEQ ID NO: 86. In one embodiment of the invention the first polypeptide comprises a first Fc region comprising a sequence as set forth in SEQ ID NO: 87. In one embodiment of the invention the first polypeptide comprises a first Fc region comprising a sequence as set forth in SEQ ID NO: 88. In one embodiment of the invention the first polypeptide comprises a first Fc region comprising a sequence as set forth in SEQ ID NO: 89. In one embodiment of the invention the first polypeptide comprises a first Fc region comprising a sequence as set forth in SEQ ID NO: 90. In one embodiment of the invention the first polypeptide comprises a first Fc region comprising a sequence as set forth in SEQ ID NO: 91. In one embodiment of the invention the first polypeptide comprises a first Fc region comprising a sequence as set forth in SEQ ID NO: 92. In one embodiment of the invention the first polypeptide comprises a first Fc region comprising a sequence as set forth in SEQ ID NO: 93. In one embodiment of the invention the first polypeptide comprises a first Fc region comprising a sequence as set forth in SEQ ID NO: 94. In one embodiment of the invention the first polypeptide comprises a first Fc region comprising a sequence as set forth in SEQ ID NO: 95. In one embodiment of the invention the first polypeptide comprises a first Fc region comprising a sequence as set forth in SEQ ID NO: 96. In one embodiment of the invention the first polypeptide comprises a first Fc region comprising a sequence as set forth in SEQ ID NO: 97. In one embodiment of the invention the first polypeptide comprises a first Fc region comprising a sequence as set forth in SEQ ID NO: 98. In one embodiment of the invention the first polypeptide comprises a first Fc region comprising a sequence as set forth in SEQ ID NO: 99. In one embodiment of the invention the first polypeptide comprises a first Fc region comprising a sequence as set forth in SEQ ID NO: 100. In one embodiment of the invention the first polypeptide comprises a first Fc region comprising a sequence as set forth in SEQ ID NO: 101. In one embodiment of the invention the first polypeptide comprises a first Fc region comprising a sequence as set forth in SEQ ID NO: 102. In one embodiment of the invention the first polypeptide comprises a first Fc region comprising a sequence as set forth in SEQ ID NO: 103. In one embodiment of the invention the first polypeptide comprises a first Fc region comprising a sequence as set forth in SEQ ID NO: 104. In one embodiment of the invention the first polypeptide comprises a first Fc region comprising a sequence as set forth in SEQ ID NO: 105. In one embodiment of the invention the first polypeptide comprises a first Fc region comprising a sequence as set forth in SEQ ID NO: 106. In one embodiment of the invention the first polypeptide comprises a first Fc region comprising a sequence as set forth in SEQ ID NO: 107. In one embodiment of the invention the first polypeptide comprises a first Fc region comprising a sequence as set forth in SEQ ID NO: 108. In one embodiment of the invention the first polypeptide comprises a first Fc region comprising a sequence as set forth in SEQ ID NO : 109. In one embodiment of the invention the first polypeptide comprises a first Fc region comprising a sequence as set forth in SEQ ID NO: 110.

In one embodiment of the invention, the second polypeptide comprises a second Fc region comprising a sequence as set forth in SEQ ID NO : 63. In one embodiment of the invention the second polypeptide comprises a second Fc region comprising a sequence as set forth in SEQ ID NO: 64. In one embodiment of the invention the second polypeptide comprises a second Fc region comprising a sequence as set forth in SEQ ID NO : 65. In one embodiment of the invention the second polypeptide comprises a second Fc region comprising a sequence as set forth in SEQ ID NO : 66. In one embodiment of the invention the second polypeptide comprises a second Fc region comprising a sequence as set forth in SEQ ID NO : 67. In one embodiment of the invention the second polypeptide comprises a second Fc region comprising a sequence as set forth in SEQ ID NO : 68. In one embodiment of the invention the second polypeptide comprises a second Fc region comprising a sequence as set forth in SEQ ID NO : 69. In one embodiment of the invention the second polypeptide comprises a second Fc region comprising a sequence as set forth in SEQ ID NO : 70. In one embodiment of the invention the second polypeptide comprises a second Fc region comprising a sequence as set forth in SEQ ID NO: 71. In one embodiment of the invention the second polypeptide comprises a second Fc region comprising a sequence as set forth in SEQ ID NO : 72. In one embodiment of the invention the second polypeptide comprises a second Fc region comprising a sequence as set forth in SEQ ID NO : 74. In one embodiment of the invention the second polypeptide comprises a second Fc region comprising a sequence as set forth in SEQ ID NO : 75. In one embodiment of the invention the second polypeptide comprises a second Fc region comprising a sequence as set forth in SEQ ID NO: 76. In one embodiment of the invention the second polypeptide comprises a second Fc region comprising a sequence as set forth in SEQ ID NO : 77. In one embodiment of the invention the second polypeptide comprises a second Fc region comprising a sequence as set forth in SEQ ID NO : 79. In one embodiment of the invention the second polypeptide comprises a second Fc region comprising a sequence as set forth in SEQ ID NO : 80. In one embodiment of the invention the second polypeptide comprises a second Fc region comprising a sequence as set forth in SEQ ID NO : 81. In one embodiment of the invention the second polypeptide comprises a second Fc region comprising a sequence as set forth in SEQ ID NO: 82. In one embodiment of the invention the second polypeptide comprises a second Fc region comprising a sequence as set forth in SEQ ID NO : 83. In one embodiment of the invention the second polypeptide comprises a second Fc region comprising a sequence as set forth in SEQ ID NO : 84. In one embodiment of the invention the second polypeptide comprises a second Fc region comprising a sequence as set forth in SEQ ID NO : 85. In one embodiment of the invention the second polypeptide comprises a second Fc region comprising a sequence as set forth in SEQ ID NO: 86. In one embodiment of the invention the second polypeptide comprises a second Fc region comprising a sequence as set forth in SEQ ID NO : 87. In one embodiment of the invention the second polypeptide comprises a second Fc region comprising a sequence as set forth in SEQ ID NO: 88. In one embodiment of the invention the second polypeptide comprises a second Fc region comprising a sequence as set forth in SEQ ID NO : 89. In one embodiment of the invention the second polypeptide comprises a second Fc region comprising a sequence as set forth in SEQ ID NO : 90. In one embodiment of the invention the second polypeptide comprises a second Fc region comprising a sequence as set forth in SEQ ID NO: 91. In one embodiment of the invention the second polypeptide comprises a second Fc region comprising a sequence as set forth in SEQ ID NO : 92. In one embodiment of the invention the second polypeptide comprises a second Fc region comprising a sequence as set forth in SEQ ID NO : 93. In one embodiment of the invention the second polypeptide comprises a second Fc region comprising a sequence as set forth in SEQ ID NO : 94. In one embodiment of the invention the second polypeptide comprises a second Fc region comprising a sequence as set forth in SEQ ID NO : 95. In one embodiment of the invention the second polypeptide comprises a second Fc region comprising a sequence as set forth in SEQ ID NO: 96. In one embodiment of the invention the second polypeptide comprises a second Fc region comprising a sequence as set forth in SEQ ID NO : 97. In one embodiment of the invention the second polypeptide comprises a second Fc region comprising a sequence as set forth in SEQ ID NO : 98. In one embodiment of the invention the second polypeptide comprises a second Fc region comprising a sequence as set forth in SEQ ID NO : 99. In one embodiment of the invention the second polypeptide comprises a second Fc region comprising a sequence as set forth in SEQ ID NO : 100. In one embodiment of the invention the second polypeptide comprises a second Fc region comprising a sequence as set forth in SEQ ID NO: 101. In one embodiment of the invention the second polypeptide comprises a second Fc region comprising a sequence as set forth in SEQ ID NO: 102. In one embodiment of the invention the second polypeptide comprises a second Fc region comprising a sequence as set forth in SEQ ID NO: 103. In one embodiment of the invention the second polypeptide comprises a second Fc region comprising a sequence as set forth in SEQ ID NO : 104. In one embodiment of the invention the second polypeptide comprises a second Fc region comprising a sequence as set forth in SEQ ID NO: 105. In one embodiment of the invention the second polypeptide comprises a second Fc region comprising a sequence as set forth in SEQ ID NO: 106. In one embodiment of the invention the second polypeptide comprises a second Fc region comprising a sequence as set forth in SEQ ID NO: 107. In one embodiment of the invention the second polypeptide comprises a second Fc region comprising a sequence as set forth in SEQ ID NO : 108. In one embodiment of the invention the second polypeptide comprises a second Fc region comprising a sequence as set forth in SEQ ID NO: 109. In one embodiment of the invention the second polypeptide comprises a second Fc region comprising a sequence as set forth in SEQ ID NO: 110.

As described above, in some embodiments of the invention further mutations may be introduced into the Fc region that alters the ability of the polypeptide or antibody to induce/mediate effector functions or other properties of the polypeptide or antibody. Such other properties may be plasma clearance and mutations relevant for such modifications are well known to persons skilled in the art.

In one embodiment of the invention, the first polypeptide comprises a first Fc region comprising a sequence selected from the group consisting of: SEQ ID NO 22, 23, 24, 25, 31, 32, and 33, wherein at most 10 mutations has been introduced. In one embodiment of the invention the second polypeptide comprises a second Fc region comprising a sequence selected from the group consisting of: SEQ ID NO 22, 23, 24, 25, 31, 32, and 33, wherein at most 10 mutations has been introduced. The at most 10 mutations introduced into said sequence may include the amino acid mutations introduced according to the present invention.

In one embodiment of the invention, the first polypeptide comprises a first Fc region comprising a sequence as set forth in : SEQ ID NO 22, wherein at most 10 mutations have been introduced . In one embodiment of the invention the first polypeptide comprises a first Fc region comprising a sequence as set forth in : SEQ ID NO 23, wherein at most 10 mutations have been introduced. In one embodiment of the invention the first polypeptide comprises a first Fc region comprising a sequence as set forth in : SEQ ID NO 24, wherein at most 10 mutations have been introduced. In one embodiment of the invention the first polypeptide comprises a first Fc region comprising a sequence as set forth in : SEQ ID NO 25, wherein at most 10 mutations have been introduced. In one embodiment of the invention the first polypeptide comprises a first Fc region comprising a sequence as set forth in : SEQ ID NO 31, wherein at most 10 mutations have been introduced. In one embodiment of the invention the first polypeptide comprises a first Fc region comprising a sequence as set forth in : SEQ ID NO 32, wherein at most 10 mutations have been introduced. In one embodiment of the invention the first polypeptide comprises a first Fc region comprising a sequence as set forth in : SEQ ID NO 32, wherein at most 10 mutations have been introduced.

In one embodiment of the invention, the second polypeptide comprises a second Fc region comprising a sequence as set forth in : SEQ ID NO 22, wherein at most 10 mutations have been introduced. In one embodiment of the invention the second polypeptide comprises a second Fc region comprising a sequence as set forth in : SEQ ID NO 23, wherein at most 10 mutations have been introduced. In one embodiment of the invention the second polypeptide comprises a second Fc region comprising a sequence as set forth in : SEQ ID NO 24, wherein at most 10 mutations have been introduced . In one embodiment of the invention the second polypeptide comprises a second Fc region comprising a sequence as set forth in : SEQ ID NO 25, wherein at most 10 mutations have been introduced. In one embodiment of the invention the second polypeptide comprises a second Fc region comprising a sequence as set forth in : SEQ ID NO 31, wherein at most 10 mutations have been introduced. In one embodiment of the invention the second polypeptide comprises a second Fc region comprising a sequence as set forth in : SEQ ID NO 32, wherein at most 10 mutations have been introduced. In one embodiment of the invention the second polypeptide comprises a second Fc region comprising a sequence as set forth in : SEQ ID NO 32, wherein at most 10 mutations have been introduced.

In one embodiment of the invention at most 10 mutations has been introduced, such as at most 9 mutations, such as at most 8 mutations, such as at most 7 mutations, such as at most 6 mutations, such as at most 5 mutations, such as at most 4 mutations, or such as at most 3 mutations.

In one embodiment of the invention the first and second polypeptide comprises a first and second Fc region comprising a sequence selected from the group consisting of: SEQ ID NO 22, 23, 24, 25, 31, 32, and 33, wherein at most 10 mutations has been introduced, such as at most 9 mutations, such as at most 8 mutations, such as at most 7 mutations, such as at most 6 mutations, such as at most 5 mutations, such as at most 4 mutations, or such as at most 3 mutations.

In one embodiment of the invention the first and second polypeptide comprises a first and second Fc region comprising the sequence as set forth in SEQ IDNO 22, wherein at most 10 mutations has been introduced, such as at most 9 mutations, such as at most 8 mutations, such as at most 7 mutations, such as at most 6 mutations, such as at most 5 mutations, such as at most 4 mutations, or such as at most 3 mutations.

In further embodiments, said first antibody is human, humanized or chimeric and/or said second antibody is human, humanized or chimeric.

The polypeptide of the invention is not limited to polypeptides, such as antibodies, which have a natural, e.g. a human Fc domain but it may also be a polypeptide having other mutations than those of the present invention, such as e.g. mutations that affect glycosylation or enable the antibody to be a bispecific antibody. By the term "natural antibody" is meant any antibody which does not comprise any genetically introduced mutations that are not naturally occurring. An antibody which comprises naturally occurring modifications, e.g. different allotypes, is thus to be understood as a "natural antibody" in the sense of the present invention, and can thereby be understood as a parent antibody. Such antibodies may serve as a template for the one or more mutations according to the present invention, and thereby providing the variant antibodies of the invention.

The polypeptide or antibody used in the invention has the specified mutations, but may also have additional mutations to introduce additional functions into the polypeptide or antibody. In one embodiment, the Fc region comprises at most ten mutations, such as nine mutations, such as eight mutations, such as seven mutations, such as six mutations, such as five mutations, such as four mutations, such as three mutations or such as two mutations. The additional mutations also allow for a variation in the Fc region at positions which are not involved in Fc-Fc interaction, as well as in positions not involved in Fc effector functions. Further, as mentioned, additional mutations may also be due to allelic variations.

Thus, in one embodiment of the invention the polypeptide or antibody has an Fc region that is an IgG lm(f), IgGlm(a), IgGlm(z), IgGlm(x) allotype or mixed allotype.

The polypeptides or antibodies used in the invention may be monospecific or multispecific, such as bispecific. Thus, in one embodiment, said first antibody is bispecific and/or said second polypeptide is bispecific.

Target antigens, target cells and diseases to be treated

As explained above, the present invention provides methods which can be used to improve the selectivity of an antibody treatment for desired target cell populations.

The methods relate to a treatment with a first and second antigen-binding polypeptide, wherein the two antibodies bind two different target antigens (a first antigen and a second antigen) and wherein the Fc regions of the antibodies have been modified such that hetero-oligomerization of the two antibodies is strongly favored over homo-oligomerization. As a result of these modifications, more antibody oligomerization will occur on cells that express both antigen targets (allowing efficient (hetero)oligomerization of the two antibodies), than on cells that only express one of the targets (resulting in inefficient or no (homo)oligomerization). As oligomerization generally enhances the efficacy of antibodies, the antibody combination treatment will be more efficacious against cells that co-express the targets than against cells that only express one of the targets. Thus, the antibody combination treatment has an improved selectivity for cells or tissues expressing both target antigens. Accordingly, by selecting two antigens that are co-expressed in a desired target cell population, but not, or less, co-expressed in cell populations that should not be targeted, a combined antibody treatment can be designed which will have a selective effect against the desired target cell populations.

Accordingly, in a preferred embodiment of the method of the invention, said first and second antigens are both cell surface-exposed molecules and ligands. Target antigens which activate, inhibit, modulate and or regulate signal transduction pathways may be particularly suitable as targets according to the present invention.

The following protein classes may also be particular suitable as antigen- binding target for the first and/or second polypeptide according to the invention, tumor necrosis receptor super family, GPI-anchored proteins, hematopoietic factor receptor family, cytokine receptor family, serine/threonine kinase receptor family, Hydrolases and regulators superfamily, hormone receptor family, B7 family-related protein, immunoglobulin superfamily, interleukin receptor family, Integrin, Ig-like cell adhesion molecule family, Protein tyrosine phosphatases, receptor type, C-type lectin, Tetraspanins, Membrane spanning 4-domains, Interleukin receptors, Activating leukocyte immunoglobulin like receptors, C-C motif chemokine receptors, G protein-coupled receptors, Toll like receptors, Receptor Tyrosine Kinases. In one embodiment of the invention the first and second antigen binding regions is capable of binding to a target antigen form the same protein class. In one embodiment of the invention the first and second antigen-binding regions is capable of binding to a target antigen from different protein classes.

In one embodiment of the invention the first antigen-binding region is capable of binding to a target antigen from the protein class of GPI-anchored proteins and the second antigen-binding region is capable of binding to a target antigen from the protein class of Tetraspanins. In one embodiment of the invention the first antigen- binding region is capable of binding to a target antigen from the protein class of Tetraspanins and the second antigen-binding region is capable of binding to a target antigen from the protein class of GPI-anchored proteins.

In one embodiment of the invention the first antigen-binding region is capable of binding to a target antigen from the protein class of GPI-anchored proteins and the second antigen-binding region is capable of binding to a target antigen from the protein class of Membrane spanning 4-domains. In one embodiment of the invention the first antigen-binding region is capable of binding to a target antigen from the protein class of Membrane spanning 4-domains and the second antigen-binding region is capable of binding to a target antigen from the protein class of GPI- anchored proteins.

In one embodiment of the invention the first antigen-binding region is capable of binding to a target antigen from the protein class of Membrane spanning 4- domains and the second antigen-binding region is capable of binding to a target antigen from the protein class of Tetraspanins. CD20 is an example of the protein class of Membrane spanning 4-domains. Example illustrates the use of the present invention on the protein class of Membrane spanning 4-domains.

CD37 is an example of the protein class of protein class of Tetraspanins. Example illustrates the use of the present invention on the protein class of Tetraspanins.

In one embodiment of the invention the first antigen-binding region is capable of binding to a target antigen from the protein class of tumor necrosis receptor super family and the second antigen-binding region is capable of binding to a target antigen from the protein class of tumor necrosis receptor super family.

In one embodiment of the invention the first antigen-binding region is capable of binding to a target antigen from the protein class of tumor necrosis receptor super family and the second antigen-binding region is capable of binding to a target antigen from the protein class of immunoglobulin superfamily.

In one embodiment of the invention the first and/or second polypeptide comprises a first antigen-binding region and/or second antigen-binding region, wherein the antigen binding region binds to a member of the tumor necrosis factor receptor super family (TNFR-SF), G-protein Coupled Receptor (GPCR) superfamily, a membrane spanning-4 domain or a membrane Tetraspanin.

Some TNFRSF are involved in apoptosis and contains an intracellular death domain such as FAS, DR4, DR5, TNFR1, DR6, DR3, EDAR and NGFR. Other TNFRSF are involved in other signal transduction pathways, such as proliferation, survival, and differentiation such as DcRl, DcR2, DcR3, OPG, TROY, XEDAR, LTbR, HVEM, TWEAK R, CD120b, 0X40, CD40, CD27, CD30, 4-1BB, RANK, TACI, BLySR, BCMA, GITR, RELT. TNF receptors are expressed in a wide variety of tissues in mammals, especially in leukocytes.

DR5 is an example of the TNFRSF class of receptors. Example 19 illustrates the use of the present invention on the TNFRSF class of receptors.

In one embodiment of the invention the first and/or second antigen-binding region binds to a member of the TNFR-SF selected form the group consisting of: FAS, DR4, DR5, TNFR1, DR6, DR3, EDAR, NGFR, 0X40, CD40, CD30, CD27, 4-1BB, RANK, TACI, BLySR, BCMA, RELT and GITR. In one embodiment of the invention the first antigen-binding region binds to DR5. In one embodiment of the invention the second antigen-binding region binds to DR5.

In one embodiment of the invention the first and/or second antigen-binding region binds to a member of the TNFR-SF which does not comprise an intracellular death domain. In one embodiment of the invention the TNFR-SF is selected from the group of: 0X40, CD40, CD30, CD27, 4-1BB, RANK, TACI, BLySR, BCMA, RELT and GITR. In one embodiment of the invention the TNFR-SF is selected form the group of: FAS, DR4, DR4, TNFR1, DR6, DR3, EDAR, and NGFR.

Polypeptides according to the invention may bind any target, examples of such targets or antigens according to the invention may be, directed against are: TNFR1, FAS, DR3, DR4, DR5, DR6, NGFR, EDAR, DcRl, DcR2, DcR3, OPG, TROY, XEDAR, LTbR, HVEM, TWEAKR, CD120b, 0X40, CD40, CD27, CD30, 4-1BB, RANK, TACI, BLySR, BCMA, GITR, RELT.

In one embodiment of the invention the first antigen-binding region binds to CAMPATH-1. In one embodiment of the invention the second antigen-binding region binds to CAMPATH-1.

In one embodiment of the invention the first antigen-binding region binds to CD20. In one embodiment of the invention the second antigen-binding region binds to CD20.

In one embodiment of the invention the first antigen-binding region binds to CD37. In one embodiment of the invention the second antigen-binding region binds to CD37.

In one embodiment of the invention the first antigen-binding region binds to CAMPATH-1 and the second antigen-binding region binds to CD20, or vice versa.

In one embodiment of the invention the first antigen-binding region binds to CD37 and the second antigen-binding region binds to CD20, or vice versa.

In a preferred embodiment of the method of the invention, said first and second antigens are co-located in cells or tissue that are target cells or target tissue for the disease or disorder to be treated. A preferred disease to be treated is cancer.

In a further preferred embodiment,

a) said first and second antigens are not co-located in cells or tissue that are not target cells or target tissue for the disease or disorder to be treated, or

b) said first and second antigens are co-located to a lesser extent in cells or tissue that are not target cells or target tissue for the disease or disorder to be treated than in cells or tissue that are target cells or target tissue for the disease or disorder to be treated.

In one embodiment of the method of the invention, said first and second antigens are not identical and are not both death receptors comprising an intracellular death domain. In another embodiment, neither the first antigen nor the second antigen is a death receptor.

It is contemplated that the increased efficacy will not only be obtained when the two target antigens are co-expressed on the same cell, but also in other situations where the target cells are in close proximity.

Dosages, modes of administration and combination therapies

The invention provides methods of treating a disease or disorder comprising administering polypeptides as defined herein to a subject in need thereof. In one embodiment, the subject is human. The method of the invention involves administering an effective amount of the polypeptides.

"Treatment" or "treating" refers to the administration of an effective amount of a therapeutically active polypeptide according to the present invention with the purpose of easing, ameliorating, arresting or eradicating (curing) symptoms or disease states.

An "effective amount" or "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result. A therapeutically effective amount of a polypeptide, such as an antibody, may vary according to factors such as the disease stage, age, sex, and weight of the individual, and the ability of the antibody to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the antibody or antibody portion are outweighed by the therapeutically beneficial effects.

Preferably, said first polypeptide and said second polypeptide are administered sequentially within a certain time interval, such as within 5 days, within 2 days, within 1 day, within 12 hours, within 6 hours, within 2 hours, within 1 hour or simultaneously. One polypeptide may be administered more frequently than the other.

Administration may be carried out by any suitable route, but will typically be parenteral, such as intravenous, intramuscular or subcutaneous. Effective dosages and the dosage regimens for the polypeptide, e.g. an antibody, depend on the disease or condition to be treated and may be determined by the persons skilled in the art. An exemplary, non-limiting range for a therapeutically effective amount of an antibody of the present invention is about 0.1 to 100 mg/kg, such as about 0.1 to 50 mg/kg, for example about 0.1 to 20 mg/kg, such as about 0.1 to 10 mg/kg, for instance about 0.5, about 0.3, about 1, about 3, about 5, or about 8 mg/kg.

The molar ratio at which the first polypeptide and the second polypeptide are administered in the method of the invention may vary depending on the target antigens to which they bind and the extent to which they are selective for the target cell population. In one embodiment of the method of the invention, said first polypeptide and said second polypeptide are administered at a 1:50 to 50:1 molar ratio, such as a 1:1 molar ratio, a 1:2 molar ratio, a 1:3 molar ratio, a 1:4 molar ratio, a 1:5 molar ratio, a 1:6 molar ratio, a 1:7 molar ratio, a 1:8 molar ratio, a 1:9 molar ratio, a 1:10 molar ratio, a 1:15 molar ratio, a 1:20 molar ratio, a 1:25 molar ratio, a 1:30 molar ratio, a 1:35 molar ratio, a 1:40 molar ratio, a 1:45 molar ratio, a 1:50 molar ratio, a 50:1 molar ratio, a 45:1 molar ratio, a 40:1 molar ratio, a 35:1 molar ratio, a 30:1 molar ratio, a 25:1 molar ratio, a 20:1 molar ratio, a 15:1 molar ratio, a 10:1 molar ratio, a 9:1 molar ratio, a 8:1 molar ratio, a 7:1 molar ratio, a 6:1 molar ratio, a 5:1 molar ratio, a 4:1 molar ratio, a 3:1 molar ratio, a 2:1 molar ratio, or an equimolar ratio.

In a preferred embodiment, said first polypeptide and said second polypeptide are administered at a 1:50 to 50:1 molar ratio, such as 1:1 molar ratio, a 1:2 molar ratio, a 1:3 molar ratio, a 1:4 molar ratio, a 1:5 molar ratio, a 1:6 molar ratio, a 1:7 molar ratio, a 1:8 molar ratio, a 1:9 molar ratio, a 1:5 molar ratio, a 1:5 molar ratio, a 1:5 molar ratio, a 1:10 molar ratio, a 1:15 molar ratio, a 1:20 molar ratio, a 1:25 molar ratio, a 1:30 molar ratio, a 1:35 molar ratio, a 1:40 molar ratio, a 1:45 molar ratio a 1:50 molar ratio, a 50:1 molar ratio, a 45:1 molar ratio, a 40:1 molar ratio, a 35:1 molar ratio, a 30:1 molar ratio a 25:1 molar ratio, a 20:1 molar ratio, a 15:1 molar ratio, a 10:1 molar ratio, a 9:1 molar ratio, a 8:1 molar ratio, a 7:1 molar ratio, a 6:1 molar ratio, a 5:1 molar ratio, a 4:1 molar ratio, a 3:1 molar ratio, a 2:1 molar ratio.

In one embodiment of the present invention the first polypeptide and the second polypeptide are administered at molar ratio of about a 1 :50 to 50: 1, such as a molar ratio of about 1:40 to 40:1, such as a molar ratio of about 1:30 to 30:1, such as a molar ratio of about 1 :20 to 20: 1, such as a molar ratio of about 1 : 10 to 10: 1, such as a molar ratio of about 1 :9 to 9: 1, such as a molar ratio of about 1 :5 to 5: 1.

Polypeptides or antibodies of the present invention may also be administered in combination therapy, i.e., combined with other therapeutic agents relevant for the disease or condition to be treated. Accordingly, in one embodiment, the antibody- containing medicament is for combination with one or more further therapeutic agents, such as cytotoxic, chemotherapeutic or anti-angiogenic agents. Such combined administration may be simultaneous, separate or sequential.

In a further embodiment, the present invention provides a method for treating or preventing disease, such as cancer, which method comprises administration to a subject in need thereof of a therapeutically effective amount of a variant or pharmaceutical composition of the present invention, in combination with radiotherapy and/or surgery.

In one embodiment of the invention the method according to any aspect or embodiment disclosed herein relates to further administering an additional therapeutic agent. In one embodiment of the invention the additional therapeutic agent is one or more anti-cancer agent(s) selected from the group consisting of chemotherapeutics (including but not limited to paclitaxel, temozolomide, cisplatin, carboplatin, oxaliplatin, irinotecan, doxorubicin, gemcitabine, 5-fluorouracil, pemetrexed), kinase inhibitors (including but not limited to sorafenib, sunitinib or everolimus), apoptosis-modulating agents (including but not limited to recombinant human TRAIL or birinapant), RAS inhibitors, proteasome inhibitors (including but not limited to bortezomib), histon deacetylase inhibitors (including but not limited to vorinostat), nutraceuticals, cytokines (including but not limited to IFN-g), antibodies or antibody mimetics (including but not limited to anti-EGFR, anti-IGF-lR, anti-VEGF, anti-CD20, anti-CD38, anti-HER2, anti-PD-1, anti-PD-Ll, anti-CTLA4, anti-CD40, anti-CD137, anti-GITR antibodies and antibody mimetics), antibody-drug conjugates.

Polypeptides

As explained above, in a further aspect, the invention relates to polypeptides that can be used in the method of the invention, in combination with a suitable "counterpart" polypeptide, so that the combination favors hetero-oligomerization over homo-oligomerization. Accordingly, the invention also relates to a polypeptide comprising a Fc region of a human IgG and an antigen-binding region capable of binding to an antigen, wherein said polypeptide comprises

a) a I253G, I253K or I253R mutation of an amino acid position corresponding to 1253 in human IgGl,

or

a H310R or H310D or mutation of an amino acid position corresponding to H310 in human IgGl,

and/or

b) a Y436N, Y436K, Y436Q or Y436R mutation of an amino acid position corresponding to Y436 in human IgGl,

or

a Q438R, Q438K, Q438H, Q438G or Q438N mutation of an amino acid position corresponding to Q438 in human IgGl,

and/or

c) a K439F, K439I, K439Y, K439T, K439V, K439W mutation of an amino acid position corresponding to K439 in human IgGl,

or

an S440K mutation of an amino acid position corresponding to S440 in human IgGl,

wherein the amino acid positions correspond to human IgGl according to EU numbering,

with the proviso that if the polypeptide comprises said S440K mutation, then at least one of the other mutations specified in options a) and b) is also present

In one embodiment, said polypeptide comprises

a) a I253G, I253K or I253R mutation of an amino acid position corresponding to 1253 in human IgGl,

or

a H310R or H310D or mutation of an amino acid position corresponding to H310 in human IgGl,

and

b) a Y436N, Y436K, Y436Q or Y436R mutation of an amino acid position corresponding to Y436 in human IgGl,

or a Q438R, Q438K, Q438H, Q438G or Q438N mutation of an amino acid position corresponding to Q438 in human IgGl.

In another embodiment, said polypeptide comprises

a) a I253G, I253K or I253R mutation of an amino acid position corresponding to 1253 in human IgGl,

or

a H310R or H310D or mutation of an amino acid position corresponding to H310 in human IgGl,

and

c) a K439F, K439I, K439Y, K439T, K439V, K439W mutation of an amino acid position corresponding to K439 in human IgGl,

or

an S440K mutation of an amino acid position corresponding to S440 in human IgGl.

In another embodiment, said polypeptide comprises

b) a Y436N, Y436K, Y436Q or Y436R mutation of an amino acid position corresponding to Y436 in human IgGl,

or

a Q438R, Q438K, Q438H, Q438G or Q438N mutation of an amino acid position corresponding to Q438 in human IgGl,

and

c) a K439F, K439I, K439Y, K439T, K439V, K439W mutation of an amino acid position corresponding to K439 in human IgGl,

or

an S440K mutation of an amino acid position corresponding to S440 in human IgGl.

In another embodiment, wherein said polypeptide comprises

a) a I253G, I253K or I253R mutation of an amino acid position corresponding to 1253 in human IgGl,

or

a H310R or H310D or mutation of an amino acid position corresponding to H310 in human IgGl, and

b) a Y436N, Y436K, Y436Q or Y436R mutation of an amino acid position corresponding to Y436 in human IgGl,

or

a Q438R, Q438K, Q438H, Q438G or Q438N mutation of an amino acid position corresponding to Q438 in human IgGl,

and

c) a K439F, K439I, K439Y, K439T, K439V, K439W mutation of an amino acid position corresponding to K439 in human IgGl,

or

an S440K mutation of an amino acid position corresponding to S440 in human IgGl.

In another embodiment, the polypeptide does not comprise the mutations specified in option c) and said polypeptide further comprises a K439E mutation of an amino acid position corresponding to K439 in human IgGl or a S440K mutation of an amino acid position corresponding to S440 in human IgGl.

In another embodiment,

i) said polypeptide comprises an I253G mutation of an amino acid position corresponding to 1253 in human IgGl

and

a K439E mutation of an amino acid position corresponding to K439 in human IgGl or a S440K mutation of an amino acid position corresponding to S440 in human IgGl,

or

ii) said polypeptide comprises an I253R mutation of an amino acid position corresponding to 1253 in human IgGl

and

a K439E mutation of an amino acid position corresponding to K439 in human IgGl or a S440K mutation of an amino acid position corresponding to S440 in human IgGl,

or

iii) said polypeptide comprises an H310R mutation of an amino acid position corresponding to H310 in human IgGl and

a K439E mutation of an amino acid position corresponding to K439 in human IgGl or a S440K mutation of an amino acid position corresponding to S440 in human IgGl,

or

iv) said polypeptide comprises an H310D mutation of an amino acid position corresponding to H310 in human IgGl

and

a K439E mutation of an amino acid position corresponding to K439 in human IgGl or a S440K mutation of an amino acid position corresponding to S440 in human IgGl,

or

v) said polypeptide comprises a Y436N mutation of an amino acid position corresponding to Y436 in human IgGl,

and

a K439E mutation of an amino acid position corresponding to K439 in human IgGl or a S440K mutation of an amino acid position corresponding to S440 in human IgGl,

or

vi) said polypeptide comprises a Y436K mutation of an amino acid position corresponding to Y436 in human IgGl,

and

a K439E mutation of an amino acid position corresponding to K439 in human IgGl or a S440K mutation of an amino acid position corresponding to S440 in human IgGl,

or

vii) said polypeptide comprises a Q438R mutation of an amino acid position corresponding to Q438 in human IgGl,

and

a K439E mutation of an amino acid position corresponding to K439 in human IgGl or a S440K mutation of an amino acid position corresponding to S440 in human IgGl,

or

viii) said polypeptide comprises a Q438N mutation of an amino acid position corresponding to Q438 in human IgGl, and

a K439E mutation of an amino acid position corresponding to K439 in human IgGl or a S440K mutation of an amino acid position corresponding to S440 in human IgGl,

or

ix) said polypeptide comprises a Y436N mutation of an amino acid position corresponding to Y436 in human IgGl,

and

a I253G mutation of an amino acid position corresponding to 1253 in human IgGl or a H310R mutation of an amino acid position corresponding to H310 in human IgGl,

or

x) said polypeptide comprises a Y436K mutation of an amino acid position corresponding to Y436 in human IgGl,

and

a I253G mutation of an amino acid position corresponding to 1253 in human IgGl or a H310R mutation of an amino acid position corresponding to H310 in human IgGl,

or

xi) said polypeptide comprises a Q438R mutation of an amino acid position corresponding to Q438 in human IgGl,

and

a I253G mutation of an amino acid position corresponding to 1253 in human IgGl or a H310R mutation of an amino acid position corresponding to H310 in human IgGl,

or

xii) said polypeptide comprises a Q438N mutation of an amino acid position corresponding to Q438 in human IgGl,

and

a I253G mutation of an amino acid position corresponding to 1253 in human IgGl or a H310R mutation of an amino acid position corresponding to H310 in human IgGl,

or

xiii) said polypeptide comprises a Y436N mutation of an amino acid position corresponding to Y436 in human IgGl, and

a I253R mutation of an amino acid position corresponding to 1253 in human IgGl or a H310D mutation of an amino acid position corresponding to H310 in human IgGl,

or

xiv) said polypeptide comprises a Y436K mutation of an amino acid position corresponding to Y436 in human IgGl,

and

a I253R mutation of an amino acid position corresponding to 1253 in human IgGl or a H310D mutation of an amino acid position corresponding to H310 in human IgGl,

or

xv) said polypeptide comprises a Q438R mutation of an amino acid position corresponding to Q438 in human IgGl,

and

a I253R mutation of an amino acid position corresponding to 1253 in human IgGl or a H310D mutation of an amino acid position corresponding to H310 in human IgGl,

or

xvi) said polypeptide comprises a Q438N mutation of an amino acid position corresponding to Q438 in human IgGl,

and

a I253R mutation of an amino acid position corresponding to 1253 in human IgGl or a H310D mutation of an amino acid position corresponding to H310 in human IgGl.

In a further embodiment, said polypeptide further comprises a mutation of an amino acid position corresponding to E430, E345, S440, T437 or K248 in human IgGl, with the proviso that if said polypeptide comprises a K439E, K439D, S440K, S440R or S440H mutation, said further mutation in said polypeptide is not at position S440.

In another embodiment, said polypeptide comprises one or more mutations selected from the group consisting of: E430G, E345K, E430S, E430F, E430T, E345Q, E345R, E345Y, S440W and S440Y and/or said polypeptide comprises a T437R and a K248E mutation. In another embodiment, said polypeptide comprises one or both mutations selected from the group consisting of: E430G and E345K.

In another embodiment, said polypeptide comprises an E430G mutation.

In another embodiment, said polypeptide has been further modified so that the polypeptide has an altered ability to induce effector functions, such as Fc- mediated effector functions, compared to a polypeptide which is identical except for said further modification.

In one such embodiment, said polypeptide has been further modified so that the polypeptide has an altered ability to induce antibody-dependent cell-mediated cytotoxicity (ADCC) compared to a polypeptide which is identical except for said further modification. An example of an amino acid mutation which alters the ability of a polypeptide or antibody to induce ADCC is G237A. A G237A mutation will decrease a polypeptides ability to bind to Fc gamma receptors and thereby decrease the polypeptides ability to induce ADCC. A polypeptide with a decrease ability to induce ADCC may be of particular interest when enhanced control of the effector functions induced by the polypeptide is of interest e.g . when the target to which the antibody binds is ubiquitously expressed. Thus, in one embodiment of the present invention said polypeptide has been modified by introducing a further G237A mutation.

In one embodiment said polypeptide comprises a G237A mutation.

In another such embodiment, said polypeptide has been further modified so that the polypeptide has an altered ability to induce complement-dependent cytotoxicity (CDC) compared to a polypeptide which is identical except for said modification.

An example of an amino acid mutations which alters the ability of a polypeptide or antibody to induce CDC are E430G, E345K, E430S, E430F, E430T, E345Q, E345R, E345Y, S440W, S440Y T437R, K248E, E333S and K326W. A polypeptide with an increased ability to induce CDC may be of particular interest when eradicating or depleting a specific cell type or tissue is of interest. Thus, in one embodiment of the present invention said polypeptide has been modified by introducing one or more amino acid mutations from the group consisting of E430G, E345K, E430S, E430F, E430T, E345Q, E345R, E345Y, S440W, S440Y T437R, K248E, E333S and K326W.

In one embodiment said polypeptide comprises an E333S and/or K326W mutation.

In one embodiment said polypeptide comprises an E333S. In one embodiment said polypeptide comprises an E333S and K326W mutation.

In one embodiment, the polypeptide is an antibody, such as a full-length antibody. In one embodiment, said polypeptide is an IgGl antibody. In one embodiment, said antibody is human, humanized or chimeric. In one embodiment, said antibody is bispecific.

In one embodiment of the polypeptide of the invention, said antigen is a cell surface-exposed molecule. In one embodiment, said antigen is not a death receptor.

The invention further relates to a pharmaceutical composition comprising a polypeptide of the invention as defined herein and a pharmaceutically-acceptable carrier.

Further aspects and embodiments of the invention

As described above, in a further aspect, the invention relates to a first polypeptide, comprising a first Fc region of a human IgG and a first antigen-binding region capable of binding to a first antigen, for use as a medicament in combination with a second polypeptide, comprising a second Fc region of a human IgG and a second antigen-binding region capable of binding to a second antigen, wherein

a) said first polypeptide comprises an I253G mutation of an amino acid position corresponding to 1253 in human IgGl and said second polypeptide comprises an H310R mutation of an amino acid position corresponding to H310 in human IgGl, or vice versa,

or

said first polypeptide comprises an I253K or I253R mutation of an amino acid position corresponding to 1253 in human IgG l and said second polypeptide comprises an H310D mutation of an amino acid position corresponding to H310 in human IgGl, or vice versa,

and/or

b) said first polypeptide comprises a Y436N, Y436K, Y436Q or Y436R mutation of an amino acid position corresponding to Y436 in human IgGl and said second polypeptide comprises a Q438R, Q438K, Q438H, Q438G or Q438N mutation of an amino acid position corresponding to Q438 in human IgGl, or vice versa, or

said first polypeptide comprises a Y436N or Y436Q mutation of an amino acid position corresponding to Y436 in human IgGl and said second polypeptide comprises a Y436K or Y436R mutation of an amino acid position corresponding to Y436 in human IgGl, or vice versa,

or

said first polypeptide comprises a Q438R, Q438K or Q438H mutation of an amino acid position corresponding to Q438 in human IgGl and said second polypeptide comprises a Q438N or Q438G mutation of an amino acid position corresponding to Q438 in human IgGl, or vice versa,

and/or

c) said first polypeptide comprises a K439F, K439I, K439Y, K439T, K439V, K439W mutation of an amino acid position corresponding to K439 in human IgGl and said second polypeptide comprises an S440K mutation of an amino acid position corresponding to S440 in human IgGl, or vice versa,

wherein the amino acid positions correspond to human IgGl according to EU numbering.

In one embodiment,

a) said first polypeptide comprises an I253G mutation of an amino acid position corresponding to 1253 in human IgGl and said second polypeptide comprises an H310R mutation of an amino acid position corresponding to H310 in human IgGl, or vice versa,

or

said first polypeptide comprises an I253K or I253R mutation of an amino acid position corresponding to 1253 in human IgGl and said second polypeptide comprises an H310D mutation of an amino acid position corresponding to H310 in human IgGl, or vice versa,

and/or

b) said first polypeptide comprises a Y436N mutation of an amino acid position corresponding to Y436 in human IgGl and said second polypeptide comprises a Q438R mutation of an amino acid position corresponding to Q438 in human IgGl, or vice versa,

or

said first polypeptide comprises a Y436N mutation of an amino acid position corresponding to Y436 in human IgGl and said second polypeptide comprises a Y436K mutation of an amino acid position corresponding to Y436 in human IgGl, or vice versa,

or said first polypeptide comprises a Q438R mutation of an amino acid position corresponding to Q438 in human IgGl and said second polypeptide comprises a Q438N mutation of an amino acid position corresponding to Q438 in human IgGl, or vice versa,

and/or

c) said first polypeptide comprises a K439F, K439I, K439Y, K439T, K439V, K439W mutation of an amino acid position corresponding to K439 in human IgGl and said second polypeptide comprises an S440K mutation of an amino acid position corresponding to S440 in human IgGl, or vice versa.

In another embodiment,

a) said first polypeptide comprises an I253G mutation of an amino acid position corresponding to 1253 in human IgGl and said second polypeptide comprises an H310R mutation of an amino acid position corresponding to H310 in human IgGl, or vice versa,

or

said first polypeptide comprises an I253K or I253R mutation of an amino acid position corresponding to 1253 in human IgGl and said second polypeptide comprises an H310D mutation of an amino acid position corresponding to H310 in human IgGl, or vice versa,

and

b) said first polypeptide comprises a Y436N, Y436K, Y436Q or Y436R mutation of an amino acid position corresponding to Y436 in human IgGl and said second polypeptide comprises a Q438R, Q438K, Q438H, Q438G or Q438N mutation of an amino acid position corresponding to Q438 in human IgGl, or vice versa, wherein preferably said first polypeptide comprises a Y436N mutation of an amino acid position corresponding to Y436 in human IgGl and said second polypeptide comprises a Q438R mutation of an amino acid position corresponding to Q438 in human IgGl, or vice versa,

or

said first polypeptide comprises a Y436N or Y436Q mutation of an amino acid position corresponding to Y436 in human IgGl and said second polypeptide comprises a Y436K or Y436R mutation of an amino acid position corresponding to Y436 in human IgGl, or vice versa, wherein preferably said first polypeptide comprises a Y436N mutation of an amino acid position corresponding to Y436 in human IgGl and said second polypeptide comprises a Y436K mutation of an amino acid position corresponding to Y436 in human IgGl, or vice versa, or

said first polypeptide comprises a Q438R, Q438K or Q438H mutation of an amino acid position corresponding to Q438 in human IgGl and said second polypeptide comprises a Q438N or Q438G mutation of an amino acid position corresponding to Q438 in human IgGl, or vice versa, wherein preferably said first polypeptide comprises a Q438R mutation of an amino acid position corresponding to Q438 in human IgGl and said second polypeptide comprises a Q438N mutation of an amino acid position corresponding to Q438 in human IgGl, or vice versa.

In another embodiment,

a) said first polypeptide comprises an I253G mutation of an amino acid position corresponding to 1253 in human IgGl and said second polypeptide comprises an H310R mutation of an amino acid position corresponding to H310 in human IgGl, or vice versa,

or

said first polypeptide comprises an I253K or I253R mutation of an amino acid position corresponding to 1253 in human IgGl and said second polypeptide comprises an H310D mutation of an amino acid position corresponding to H310 in human IgGl, or vice versa,

and

c) said first polypeptide comprises a K439F, K439I, K439Y, K439T, K439V, K439W mutation of an amino acid position corresponding to K439 in human IgGl and said second polypeptide comprises an S440K mutation of an amino acid position corresponding to S440 in human IgGl, or vice versa.

In another embodiment,

b) said first polypeptide comprises a Y436N, Y436K, Y436Q or Y436R mutation of an amino acid position corresponding to Y436 in human IgGl and said second polypeptide comprises a Q438R, Q438K, Q438H, Q438G or Q438N mutation of an amino acid position corresponding to Q438 in human IgGl, or vice versa, wherein preferably said first polypeptide comprises a Y436N mutation of an amino acid position corresponding to Y436 in human IgGl and said second polypeptide comprises a Q438R mutation of an amino acid position corresponding to Q438 in human IgGl, or vice versa,

or said first polypeptide comprises a Y436N or Y436Q mutation of an amino acid position corresponding to Y436 in human IgGl and said second polypeptide comprises a Y436K or Y436R mutation of an amino acid position corresponding to Y436 in human IgGl, or vice versa, wherein preferably said first polypeptide comprises a Y436N mutation of an amino acid position corresponding to Y436 in human IgGl and said second polypeptide comprises a Y436K mutation of an amino acid position corresponding to Y436 in human IgGl, or vice versa, or

said first polypeptide comprises a Q438R, Q438K or Q438H mutation of an amino acid position corresponding to Q438 in human IgGl and said second polypeptide comprises a Q438N or Q438G mutation of an amino acid position corresponding to Q438 in human IgGl, or vice versa, wherein preferably said first polypeptide comprises a Q438R mutation of an amino acid position corresponding to Q438 in human IgGl and said second polypeptide comprises a Q438N mutation of an amino acid position corresponding to Q438 in human IgGl, or vice versa and

c) said first polypeptide comprises a K439F, K439I, K439Y, K439T, K439V, K439W mutation of an amino acid position corresponding to K439 in human IgGl and said second polypeptide comprises an S440K mutation of an amino acid position corresponding to S440 in human IgGl, or vice versa.

In another embodiment,

a) said first polypeptide comprises an I253G mutation of an amino acid position corresponding to 1253 in human IgGl and said second polypeptide comprises an H310R mutation of an amino acid position corresponding to H310 in human IgGl, or vice versa,

or

said first polypeptide comprises an I253K or I253R mutation of an amino acid position corresponding to 1253 in human IgGl and said second polypeptide comprises an H310D mutation of an amino acid position corresponding to H310 in human IgGl, or vice versa,

and

b) said first polypeptide comprises a Y436N, Y436K, Y436Q or Y436R mutation of an amino acid position corresponding to Y436 in human IgGl and said second polypeptide comprises a Q438R, Q438K, Q438H, Q438G or Q438N mutation of an amino acid position corresponding to Q438 in human IgGl, or vice versa, wherein preferably said first polypeptide comprises a Y436N mutation of an amino acid position corresponding to Y436 in human IgGl and said second polypeptide comprises a Q438R mutation of an amino acid position corresponding to Q438 in human IgGl, or vice versa,

or

said first polypeptide comprises a Y436N or Y436Q mutation of an amino acid position corresponding to Y436 in human IgGl and said second polypeptide comprises a Y436K or Y436R mutation of an amino acid position corresponding to Y436 in human IgGl, or vice versa, wherein preferably said first polypeptide comprises a Y436N mutation of an amino acid position corresponding to Y436 in human IgGl and said second polypeptide comprises a Y436K mutation of an amino acid position corresponding to Y436 in human IgGl, or vice versa, or

said first polypeptide comprises a Q438R, Q438K or Q438H mutation of an amino acid position corresponding to Q438 in human IgGl and said second polypeptide comprises a Q438N or Q438G mutation of an amino acid position corresponding to Q438 in human IgGl, or vice versa, wherein preferably said first polypeptide comprises a Q438R mutation of an amino acid position corresponding to Q438 in human IgGl and said second polypeptide comprises a Q438N mutation of an amino acid position corresponding to Q438 in human IgGl, or vice versa, and

c) said first polypeptide comprises a K439F, K439I, K439Y, K439T, K439V, K439W mutation of an amino acid position corresponding to K439 in human IgGl and said second polypeptide comprises an S440K mutation of an amino acid position corresponding to S440 in human IgGl, or vice versa.

In a further embodiment, the first and second polypeptides do not comprise the mutations specified in option c) and said first polypeptide further comprises a K439E mutation of an amino acid position corresponding to K439 in human IgGl and said second polypeptide further comprises an S440K mutation of an amino acid position corresponding to S440 in human IgGl, or vice versa.

In a further embodiment,

i) said first polypeptide comprises an I253G mutation of an amino acid position corresponding to 1253 in human IgGl and said second polypeptide comprises an H310R mutation of an amino acid position corresponding to H310 in human IgGl, or vice versa,

or

ii) said first polypeptide comprises an I253R mutation of an amino acid position corresponding to 1253 in human IgGl and said second polypeptide comprises an H310D mutation of an amino acid position corresponding to H310 in human IgGl, or vice versa,

or

iii) said first polypeptide comprises an I253G mutation of an amino acid position corresponding to 1253 in human IgGl and said second polypeptide comprises an H310R mutation of an amino acid position corresponding to H310 in human IgGl, or vice versa, and said first polypeptide comprises a K439E mutation of an amino acid position corresponding to K439 in human IgGl and said second polypeptide comprises an S440K mutation of an amino acid position corresponding to S440 in human IgGl, or vice versa,

or

iv) said first polypeptide comprises an I253R mutation of an amino acid position corresponding to 1253 in human IgGl and said second polypeptide comprises an H310D mutation of an amino acid position corresponding to H310 in human IgGl, or vice versa, and said first polypeptide comprises a K439E mutation of an amino acid position corresponding to K439 in human IgGl and said second polypeptide comprises an S440K mutation of an amino acid position corresponding to S440 in human IgGl, or vice versa,

or

v) said first polypeptide comprises a Y436N mutation of an amino acid position corresponding to Y436 in human IgGl and said second polypeptide comprises a Q438R mutation of an amino acid position corresponding to Q438 in human IgGl, or vice versa,

or

vi) said first polypeptide comprises a Y436N mutation of an amino acid position corresponding to Y436 in human IgGl and said second polypeptide comprises a Y436K mutation of an amino acid position corresponding to Y436 in human IgGl, or vice versa,

or vii) said first polypeptide comprises a Q438R mutation of an amino acid position corresponding to Q438 in human IgGl and said second polypeptide comprises a Q438N mutation of an amino acid position corresponding to Q438 in human IgGl, or vice versa,

or

viii) said first polypeptide comprises a Y436N mutation of an amino acid position corresponding to Y436 in human IgGl and said second polypeptide comprises a Q438R mutation of an amino acid position corresponding to Q438 in human IgGl, or vice versa, and said first polypeptide comprises an I253G mutation of an amino acid position corresponding to 1253 in human IgGl and said second polypeptide comprises an H310R mutation of an amino acid position corresponding to H310 in human IgGl, or vice versa,

or

ix) said first polypeptide comprises a Y436N mutation of an amino acid position corresponding to Y436 in human IgGl and said second polypeptide comprises a Q438R mutation of an amino acid position corresponding to Q438 in human IgGl, or vice versa, said first polypeptide comprises an I253R mutation of an amino acid position corresponding to 1253 in human IgGl and said second polypeptide comprises an H310D mutation of an amino acid position corresponding to H310 in human IgGl, or vice versa,

or

x) said first polypeptide comprises a Y436N mutation of an amino acid position corresponding to Y436 in human IgGl and said second polypeptide comprises a Q438R mutation of an amino acid position corresponding to Q438 in human IgGl, or vice versa, and said first polypeptide comprises an I253G mutation of an amino acid position corresponding to 1253 in human IgGl and said second polypeptide comprises an H310R mutation of an amino acid position corresponding to H310 in human IgGl, or vice versa, and said first polypeptide comprises a K439E mutation of an amino acid position corresponding to K439 in human IgGl and said second polypeptide comprises an S440K mutation of an amino acid position corresponding to S440 in human IgGl, or vice versa,

or

xi) said first polypeptide comprises a Y436N mutation of an amino acid position corresponding to Y436 in human IgGl and said second polypeptide comprises a Q438R mutation of an amino acid position corresponding to Q438 in human IgGl, or vice versa, and said first polypeptide comprises an I253R mutation of an amino acid position corresponding to 1253 in human IgGl and said second polypeptide comprises an H310D mutation of an amino acid position corresponding to H310 in human IgGl, or vice versa, and said first polypeptide comprises a K439E mutation of an amino acid position corresponding to K439 in human IgGl and said second polypeptide comprises an S440K mutation of an amino acid position corresponding to S440 in human IgGl, or vice versa,

or

xii) said first polypeptide comprises a Y436N mutation of an amino acid position corresponding to Y436 in human IgGl and said second polypeptide comprises a Y436K mutation of an amino acid position corresponding to Y436 in human IgGl, or vice versa, said first polypeptide comprises an I253G mutation of an amino acid position corresponding to 1253 in human IgGl and said second polypeptide comprises an H310R mutation of an amino acid position corresponding to H310 in human IgGl, or vice versa,

or

xiii) said first polypeptide comprises a Y436N mutation of an amino acid position corresponding to Y436 in human IgGl and said second polypeptide comprises a Y436K mutation of an amino acid position corresponding to Y436 in human IgGl, or vice versa, said first polypeptide comprises an I253R mutation of an amino acid position corresponding to 1253 in human IgGl and said second polypeptide comprises an H310D mutation of an amino acid position corresponding to H310 in human IgGl, or vice versa,

or

xiv) said first polypeptide comprises a Y436N mutation of an amino acid position corresponding to Y436 in human IgGl and said second polypeptide comprises a Y436K mutation of an amino acid position corresponding to Y436 in human IgGl, or vice versa, said first polypeptide comprises an I253G mutation of an amino acid position corresponding to 1253 in human IgGl and said second polypeptide comprises an H310R mutation of an amino acid position corresponding to H310 in human IgGl, or vice versa, and said first polypeptide comprises a K439E mutation of an amino acid position corresponding to K439 in human IgGl and said second polypeptide comprises an S440K mutation of an amino acid position corresponding to S440 in human IgGl, or vice versa,

or xv) said first polypeptide comprises a Y436N mutation of an amino acid position corresponding to Y436 in human IgGl and said second polypeptide comprises a Y436K mutation of an amino acid position corresponding to Y436 in human IgGl, or vice versa, said first polypeptide comprises an I253R mutation of an amino acid position corresponding to 1253 in human IgGl and said second polypeptide comprises an H310D mutation of an amino acid position corresponding to H310 in human IgGl, or vice versa, and said first polypeptide comprises a K439E mutation of an amino acid position corresponding to K439 in human IgGl and said second polypeptide comprises an S440K mutation of an amino acid position corresponding to S440 in human IgGl, or vice versa,

or

xvi) said first polypeptide comprises a Q438R mutation of an amino acid position corresponding to Q438 in human IgGl and said second polypeptide comprises a Q438N mutation of an amino acid position corresponding to Q438 in human IgGl, or vice versa, said first polypeptide comprises an I253G mutation of an amino acid position corresponding to 1253 in human IgGl and said second polypeptide comprises an H310R mutation of an amino acid position corresponding to H310 in human IgGl, or vice versa,

or

xvii) said first polypeptide comprises a Q438R mutation of an amino acid position corresponding to Q438 in human IgGl and said second polypeptide comprises a Q438N mutation of an amino acid position corresponding to Q438 in human IgGl, or vice versa, said first polypeptide comprises an I253R mutation of an amino acid position corresponding to 1253 in human IgGl and said second polypeptide comprises an H310D mutation of an amino acid position corresponding to H310 in human IgGl, or vice versa,

or

xviii) said first polypeptide comprises a Q438R mutation of an amino acid position corresponding to Q438 in human IgGl and said second polypeptide comprises a Q438N mutation of an amino acid position corresponding to Q438 in human IgGl, or vice versa, said first polypeptide comprises an I253G mutation of an amino acid position corresponding to 1253 in human IgGl and said second polypeptide comprises an H310R mutation of an amino acid position corresponding to H310 in human IgGl, or vice versa, and said first polypeptide comprises a K439E mutation of an amino acid position corresponding to K439 in human IgGl and said second polypeptide comprises an S440K mutation of an amino acid position corresponding to S440 in human IgGl, or vice versa,

or

xix) said first polypeptide comprises a Q438R mutation of an amino acid position corresponding to Q438 in human IgGl and said second polypeptide comprises a Q438N mutation of an amino acid position corresponding to Q438 in human IgGl, or vice versa, said first polypeptide comprises an I253R mutation of an amino acid position corresponding to 1253 in human IgGl and said second polypeptide comprises an H310D mutation of an amino acid position corresponding to H310 in human IgGl, or vice versa, and said first polypeptide comprises a K439E mutation of an amino acid position corresponding to K439 in human IgGl and said second polypeptide comprises an S440K mutation of an amino acid position corresponding to S440 in human IgGl, or vice versa,

or

xx) said first polypeptide comprises a Y436N mutation of an amino acid position corresponding to Y436 in human IgGl and said second polypeptide comprises a Q438R mutation of an amino acid position corresponding to Q438 in human IgGl, or vice versa, and said first polypeptide comprises a K439E mutation of an amino acid position corresponding to K439 in human IgGl and said second polypeptide comprises an S440K mutation of an amino acid position corresponding to S440 in human IgGl, or vice versa,

or

xxi) said first polypeptide comprises a Y436N mutation of an amino acid position corresponding to Y436 in human IgGl and said second polypeptide comprises a Y436K mutation of an amino acid position corresponding to Y436 in human IgGl, or vice versa, and said first polypeptide comprises a K439E mutation of an amino acid position corresponding to K439 in human IgGl and said second polypeptide comprises an S440K mutation of an amino acid position corresponding to S440 in human IgGl, or vice versa,

or

xxii) said first polypeptide comprises a Q438R mutation of an amino acid position corresponding to Q438 in human IgGl and said second polypeptide comprises a Q438N mutation of an amino acid position corresponding to Q438 in human IgGl, or vice versa, and said first polypeptide comprises a K439E mutation of an amino acid position corresponding to K439 in human IgGl and said second polypeptide comprises an S440K mutation of an amino acid position corresponding to S440 in human IgGl, or vice versa.

In a further embodiment, said first polypeptide further comprises a mutation of an amino acid position corresponding to E430, E345, S440, T437 or K248 in human IgGl, and/or said second polypeptide further comprises a mutation of an amino acid position corresponding to E430, E345, S440, T437 or K248 in human IgGl, or vice versa,

with the proviso that if said first or second polypeptide comprises a K439E, K439D, S440K, S440R or S440H mutation, said further mutation in said polypeptide is not at position S440.

In a further embodiment hereof, said first polypeptide comprises one or more mutations selected from the group consisting of: E430G, E345K, E430S, E430F, E430T, E345Q, E345R, E345Y, S440W and S440Y, and/or said second polypeptide comprises one or more mutations selected from the group consisting of: E430G, E345K, E430S, E430F, E430T, E345Q, E345R, E345Y, S440W and S440Y,

and/or

said first polypeptide comprises a T437R and a K248E mutation, and/or said second polypeptide comprises a T437R and a K248E mutation.

In an even further embodiment hereof, said first polypeptide comprises one or both mutations selected from the group consisting of: E430G and E345K, and/or said polypeptide comprises one or both mutations selected from the group consisting of: E430G and E345K.

In a yet even further embodiment hereof, said first polypeptide comprises E430G and said second polypeptide comprises E430G.

In another embodiment, said first polypeptide and/or said second polypeptide has been further modified so that the polypeptide has an altered ability to induce effector functions, such as Fc-mediated effector functions, compared to a polypeptide which is identical except for said further modification.

In one embodiment, said first polypeptide and/or said second polypeptide has been further modified so that the polypeptide has an altered ability to induce antibody-dependent cell-mediated cytotoxicity compared to a polypeptide which is identical except for said further modification.

In another embodiment, said first polypeptide and/or said second polypeptide has been further modified so that the polypeptide has an altered ability to induce complement-dependent cytotoxicity compared to a polypeptide which is identical except for said modification.

In one embodiment, said first polypeptide is an antibody, such as a full-length antibody and/or said second polypeptide is an antibody, such as a full-length antibody.

In one embodiment, said first polypeptide is an IgGl antibody and/or said second polypeptide is an IgGl antibody.

In one embodiment, said first antibody is human, humanized or chimeric and/or said second antibody is human, humanized or chimeric.

In one embodiment, said first antibody is bispecific and/or said second polypeptide is bispecific.

In one embodiment, said first and second antigens are both cell surface- exposed molecules.

In one embodiment, said first and second antigens are co-located in cells or tissues that are target cells or target tissue for the disease or disorder to be treated. In a further embodiment,

a) said first and second antigens are not co-located in cells or tissue that are not target cells or target tissue for the disease or disorder to be treated, or

b) said first and second antigens are co-located to a lesser extent in cells or tissue that are not target cells or target tissue for the disease or disorder to be treated than in cells or tissue that are target cells or target tissue for the disease or disorder to be treated.

In one embodiment, said first and second antigens are not identical and are not both death receptors comprising an intracellular death domain. In a further embodiment, neither the first antigen nor the second antigen is a death receptor.

In one embodiment, said first polypeptide and said second polypeptide are administered at a 1:50 to 50:1 molar ratio, such as 1:1 molar ratio, a 1:2 molar ratio, a 1:3 molar ratio, a 1:4 molar ratio, a 1:5 molar ratio, a 1:6 molar ratio, a 1:7 molar ratio, a 1:8 molar ratio, a 1:9 molar ratio, a 1:5 molar ratio, a 1:5 molar ratio, a 1:5 molar ratio, a 1:10 molar ratio, a 1:15 molar ratio, a 1:20 molar ratio, a 1:25 molar ratio, a 1:30 molar ratio, a 1:35 molar ratio, a 1:40 molar ratio, a 1:45 molar ratio a 1:50 molar ratio, a 50:1 molar ratio, a 45:1 molar ratio, a 40:1 molar ratio, a 35:1 molar ratio, a 30:1 molar ratio a 25:1 molar ratio, a 20:1 molar ratio, a 15:1 molar ratio, a 10:1 molar ratio, a 9:1 molar ratio, a 8:1 molar ratio, a 7:1 molar ratio, a 6: 1 molar ratio, a 5 : 1 molar ratio, a 4: 1 molar ratio, a 3 : 1 molar ratio, a 2 : 1 molar ratio.

In one embodiment of the present invention said first polypeptide and said second polypeptide are administered at molar ratio of about a 1 : 50 to 50 : 1, such as a molar ratio of about 1 :40 to 40 : 1, such as a molar ratio of about 1 :30 to 30 : 1, such as a molar ratio of about 1 : 20 to 20: 1, such as a molar ratio of about 1 : 10 to 10 : 1, such as a molar ratio of about 1 :9 to 9 : 1, such as a molar ratio of about 1 : 5 to 5: 1.

In one embodiment, said first polypeptide and said second polypeptide are administered simultaneously.

In one embodiment of the invention, said first polypeptide and said second polypeptide are administered simultaneously.

In one embodiment, the use is for the treatment of cancer.

In an even further aspect, the invention relates to the use of a first polypeptide, comprising a first Fc region of a human IgG and a first antigen-binding region capable of binding to a first antigen, in combination with a second polypeptide, comprising a second Fc region of a human IgG and a second antigen- binding region capable of binding to a second antigen, for the manufacture of a medicament for the treatment of cancer, wherein

a) said first polypeptide comprises an I253G mutation of an amino acid position corresponding to 1253 in human IgGl and said second polypeptide comprises an H310R mutation of an amino acid position corresponding to H310 in human IgGl, or vice versa,

or

said first polypeptide comprises an I253K or I253R mutation of an amino acid position corresponding to 1253 in human IgGl and said second polypeptide comprises an H310D mutation of an amino acid position corresponding to H310 in human IgGl, or vice versa,

and/or

b) said first polypeptide comprises a Y436N, Y436K, Y436Q or Y436R mutation of an amino acid position corresponding to Y436 in human IgGl and said second polypeptide comprises a Q438R, Q438K, Q438H, Q438G or Q438N mutation of an amino acid position corresponding to Q438 in human IgGl, or vice versa, or said first polypeptide comprises a Y436N or Y436Q mutation of an amino acid position corresponding to Y436 in human IgGl and said second polypeptide comprises a Y436K or Y436R mutation of an amino acid position corresponding to Y436 in human IgGl, or vice versa,

or

said first polypeptide comprises a Q438R, Q438K or Q438H mutation of an amino acid position corresponding to Q438 in human IgGl and said second polypeptide comprises a Q438N or Q438G mutation of an amino acid position corresponding to Q438 in human IgGl, or vice versa,

and/or

c) said first polypeptide comprises a K439F, K439I, K439Y, K439T, K439V, K439W mutation of an amino acid position corresponding to K439 in human IgGl and said second polypeptide comprises an S440K mutation of an amino acid position corresponding to S440 in human IgGl, or vice versa,

wherein the amino acid positions correspond to human IgGl according to EU numbering.

Compositions

As described above, in some embodiments of the method of the invention, the first and second polypeptides are administered separately. In other embodiments, however, the polypeptides may be formulated together in one pharmaceutical composition.

In a main aspect, the invention relates to a composition comprising a first polypeptide and a second polypeptide as defined herein.

Accordingly, the invention relates to a composition comprising a first polypeptide comprising a first Fc region of a human IgG and a first antigen-binding region capable of binding to a first antigen, in combination with a second polypeptide comprising a second Fc region of a human IgG and a second antigen-binding region capable of binding to a second antigen, wherein

d) said first polypeptide comprises an I253G mutation of an amino acid position corresponding to 1253 in human IgGl and said second polypeptide comprises an H310R mutation of an amino acid position corresponding to H310 in human IgGl, or vice versa,

or said first polypeptide comprises an I253K or I253R mutation of an amino acid position corresponding to 1253 in human IgGl and said second polypeptide comprises an H310D mutation of an amino acid position corresponding to H310 in human IgGl, or vice versa,

and/or

e) said first polypeptide comprises a Y436N, Y436K, Y436Q or Y436R mutation of an amino acid position corresponding to Y436 in human IgGl and said second polypeptide comprises a Q438R, Q438K, Q438H, Q438G or Q438N mutation of an amino acid position corresponding to Q438 in human IgGl, or vice versa, or

said first polypeptide comprises a Y436N or Y436Q mutation of an amino acid position corresponding to Y436 in human IgGl and said second polypeptide comprises a Y436K or Y436R mutation of an amino acid position corresponding to Y436 in human IgGl, or vice versa,

or

said first polypeptide comprises a Q438R, Q438K or Q438H mutation of an amino acid position corresponding to Q438 in human IgGl and said second polypeptide comprises a Q438N or Q438G mutation of an amino acid position corresponding to Q438 in human IgGl, or vice versa,

and/or

f) said first polypeptide comprises a K439F, K439I, K439Y, K439T, K439V, K439W mutation of an amino acid position corresponding to K439 in human IgGl and said second polypeptide comprises an S440K mutation of an amino acid position corresponding to S440 in human IgGl, or vice versa,

wherein the amino acid positions correspond to human IgGl according to EU numbering.

In one embodiment of the invention, said first polypeptide comprises a Y436N, Y436K, Y436Q or Y436R mutation of an amino acid position corresponding to Y436 in human IgGl and said second polypeptide comprises a Q438R, Q438K, Q438H, Q438G or Q438N mutation of an amino acid position corresponding to Q438 in human IgGl, or vice versa,

or

said first polypeptide comprises a Y436N or Y436Q mutation of an amino acid position corresponding to Y436 in human IgGl and said second polypeptide comprises a Y436K or Y436R mutation of an amino acid position corresponding to Y436 in human IgGl, or vice versa,

or

said first polypeptide comprises a Q438R, Q438K or Q438H mutation of an amino acid position corresponding to Q438 in human IgGl and said second polypeptide comprises a Q438N or Q438G mutation of an amino acid position corresponding to Q438 in human IgGl, or vice versa,

wherein the amino acid positions correspond to human IgGl according to EU numbering.

In one embodiment of the invention, said first polypeptide comprises a Y436N, Y436K, Y436Q or Y436R mutation of an amino acid position corresponding to Y436 in human IgGl and said second polypeptide comprises a Q438R, Q438K, Q438H, Q438G or Q438N mutation of an amino acid position corresponding to Q438 in human IgGl, or vice versa,

wherein the amino acid positions correspond to human IgGl according to EU numbering.

In one embodiment of the invention, said first polypeptide comprises a Y436N or Y436K, mutation of an amino acid position corresponding to Y436 in human IgGl and said second polypeptide comprises a Q438R or Q438N mutation of an amino acid position corresponding to Q438 in human IgGl, or vice versa,

or

said first polypeptide comprises a Q438R mutation of an amino acid position corresponding to Q438 in human IgGl and said second polypeptide comprises a Q438N mutation of an amino acid position corresponding to Q438 in human IgGl, or vice versa,

wherein the amino acid positions correspond to human IgGl according to EU numbering.

In one embodiment of the invention, said first polypeptide comprises a Y436N or Y436K mutation of an amino acid position corresponding to Q436 in human IgGl and said second polypeptide comprises a Q438N or Q438R mutation of an amino acid position corresponding to Q438 in human IgGl, or vice versa.

In one embodiment of the invention, said first polypeptide comprises a Y436N mutation of an amino acid position corresponding to Q436 in human IgGl and said second polypeptide comprises a Q438R mutation of an amino acid position corresponding to Q438 in human IgGl, or vice versa. Thus, in a further main aspect, the invention relates to a pharmaceutical composition comprising a first polypeptide and a second polypeptide as defined herein and a pharmaceutically-acceptable carrier.

In one embodiment, said first polypeptide and said second polypeptide are present in the composition at a 1: 50 to 50: 1 molar ratio, such as a 1 : 1 molar ratio, a 1: 2 molar ratio, a 1 :3 molar ratio, a 1:4 molar ratio, a 1 : 5 molar ratio, a 1 :6 molar ratio, a 1 :7 molar ratio, a 1 :8 molar ratio, a 1 :9 molar ratio, a 1 : 10 molar ratio, a 1: 15 molar ratio, a 1 :20 molar ratio, a 1 :25 molar ratio, a 1: 30 molar ratio, a 1 :35 molar ratio, a 1 :40 molar ratio, a 1 :45 molar ratio, a 1 : 50 molar ratio, a 50: 1 molar ratio, a 45 : 1 molar ratio, a 40 : 1 molar ratio, a 35: 1 molar ratio, a 30: 1 molar ratio, a 25: 1 molar ratio, a 20: 1 molar ratio, a 15 : 1 molar ratio, a 10: 1 molar ratio, a 9: 1 molar ratio, a 8: 1 molar ratio, a 7: 1 molar ratio, a 6: 1 molar ratio, a 5: 1 molar ratio, a 4: 1 molar ratio, a 3: 1 molar ratio, a 2: 1 molar ratio, or an equimolar ratio.

In one embodiment of the present invention said first polypeptide and said second polypeptide are present in the composition at molar ratio of about a 1 : 50 to 50: 1, such as a molar ratio of about 1 :40 to 40: 1, such as a molar ratio of about 1: 30 to 30: 1, such as a molar ratio of about 1 :20 to 20: 1, such as a molar ratio of about 1 : 10 to 10: 1, such as a molar ratio of about 1 :9 to 9: 1, such as a molar ratio of about 1 :5 to 5: 1.

In one embodiment of the present invention said first polypeptide and said second polypeptide are present in the composition at molar ratio of about a 1 : 1.

Polypeptides for use according to the invention may be formulated with pharmaceutically acceptable carriers or diluents as well as any other known adjuvants and excipients in accordance with conventional techniques such as those disclosed in (Rowe et al., Handbook of Pharmaceutical Excipients, 2012 June, ISBN 9780857110275). The pharmaceutically acceptable carriers or diluents as well as any other known adjuvants and excipients should be suitable for the polypeptides or antibodies and the chosen mode of administration. Suitability for carriers and other components of pharmaceutical compositions is determined based on the lack of significant negative impact on the desired biological properties of the chosen compound or pharmaceutical composition of the present invention (e.g., less than a substantial impact (10% or less relative inhibition, 5% or less relative inhibition, etc.) upon antigen binding). A pharmaceutical composition may also include diluents, fillers, salts, buffers, detergents (e.g., a nonionic detergent, such as Tween-20 or Tween-80), stabilizers (e.g., sugars or protein-free amino acids), preservatives, tissue fixatives, solubilizers, and/or other materials suitable for inclusion in a pharmaceutical composition.

In one embodiment of the present invention the pharmaceutical composition comprises polypeptides together with a pharmaceutical carrier. Pharmaceutically acceptable carriers include any and all suitable solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonicity agents, antioxidants and absorption- delaying agents, and the like that are physiologically compatible with a compound of the present invention.

Examples of suitable aqueous and non-aqueous carriers which may be employed in the pharmaceutical compositions of the present invention include water, saline, phosphate-buffered saline, ethanol, dextrose, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, corn oil, peanut oil, cottonseed oil, and sesame oil, carboxymethyl cellulose colloidal solutions, tragacanth gum and injectable organic esters, such as ethyl oleate, and/or various buffers. Other carriers are well known in the pharmaceutical arts.

Pharmaceutical compositions of the present invention may also comprise pharmaceutically acceptable antioxidants for instance (1) water-soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium meta bisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal-chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

Pharmaceutical compositions of the present invention may also comprise isotonicity agents, such as sugars, polyalcohols, such as mannitol, sorbitol, glycerol or sodium chloride in the compositions.

The pharmaceutical compositions of the present invention may also contain one or more adjuvants appropriate for the chosen route of administration such as preservatives, wetting agents, emulsifying agents, dispersing agents, preservatives or buffers, which may enhance the shelf life or effectiveness of the pharmaceutical composition. The compounds of the present invention may be prepared with carriers that will protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and micro-encapsulated delivery systems. Such carriers may include gelatin, glyceryl monostearate, glyceryl distearate, biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, poly-ortho-esters, and polylactic acid alone or with a wax, or other materials well known in the art. Methods for the preparation of such formulations are generally known to those skilled in the art.

The actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present invention employed, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

Kit-of-parts

The invention also relates to kit-of-parts for simultaneous, separate or sequential use in therapy comprising polypeptides or antibodies described herein.

Thus, in a further aspect, the invention relates to a kit, i.e. a kit-of-parts, comprising a first container comprising a first polypeptide of the invention as defined herein and a second container comprising a second polypeptide of the invention as defined herein.

In a further aspect, the invention relates to a device, such as a dual chamber syringe, comprising a first compartment comprising a first polypeptide according to the invention as defined herein and a second compartment comprising a second polypeptide of the invention as defined herein. In one embodiment, the device is an administration device, such as a dual chamber syringe, i.e. a syringe comprising two compartments, one compartment comprising the first polypeptide and a second compartment comprising the second polypeptide. Conjugates

In one embodiment, the first and/or second polypeptide or antibody used in the invention is conjugated, optionally via a linker, to one or more therapeutic moieties, such as a cytotoxin, a chemotherapeutic drug, a cytokine, an immunosuppressant, and/or a radioisotope. Such conjugates are referred to herein as "immunoconjugates" or "drug conjugates". Immunoconjugates which include one or more cytotoxins are referred to as "immunotoxins".

A cytotoxin or cytotoxic agent includes any agent that is detrimental to (e.g., kills) cells. Suitable therapeutic agents for forming immunoconjugates of the present invention include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, maytansine or an analog or derivative thereof, enediyene antitumor antibiotics including neocarzinostatin, calicheamycins, esperamicins, dynemicins, lidamycin, kedarcidin or analogs or derivatives thereof, anthracyclins, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, antimetabolites (such as methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, fludarabin, 5-fluorouracil, decarbazine, hydroxyurea, asparaginase, gemcitabine, cladribine), alkylating agents (such as mechlorethamine, thioepa, chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, dacarbazine (DTIC), procarbazine, mitomycin C, cisplatin and other platinum derivatives, such as carboplatin; as well as duocarmycin A, duocarmycin SA, CC-1065 (a.k.a. rachelmycin), or analogs or derivatives of CC-1065), dolastatin, pyrrolo[2,l-c][l,4] benzodiazepins (PDBs) or analogues thereof, antibiotics (such as dactinomycin (formerly actinomycin), bleomycin, daunorubicin (formerly daunomycin), doxorubicin, idarubicin, mithramycin, mitomycin, mitoxantrone, plicamycin, anthramycin (AMC)), anti-mitotic agents (e.g. , tubulin-inhibitors) such as monomethyl auristatin E, monomethyl auristatin F, or other analogs or derivatives of dolastatin 10; Histone deacetylase inhibitors such as the hydroxamic acids trichostatin A, vorinostat (SAHA), belinostat, LAQ824, and panobinostat as well as the benzamides, entinostat, CI994, mocetinostat and aliphatic acid compounds such as phenylbutyrate and valproic acid, proteasome inhibitors such as Danoprevir, bortezomib, amatoxins such as alpha- amantin, diphtheria toxin and related molecules (such as diphtheria A chain and active fragments thereof and hybrid molecules); ricin toxin (such as ricin A or a deglycosylated ricin A chain toxin), cholera toxin, a Shiga-like toxin (SLT-I, SLT-II, SLT-IIV), LT toxin, C3 toxin, Shiga toxin, pertussis toxin, tetanus toxin, soybean Bowman-Birk protease inhibitor, Pseudomonas exotoxin, alorin, saporin, modeccin, gelanin, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, and enomycin toxins. Other suitable conjugated molecules include antimicrobial/lytic peptides such as CLIP, Magainin 2, mellitin, Cecropin, and P18; ribonuclease (RNase), DNase I, Staphylococcal enterotoxin-A, pokeweed antiviral protein, diphtherin toxin, and Pseudomonas endotoxin. See, for example, Pastan et al., Cell 47, 641 (1986) and Goldenberg, Calif. A Cancer Journal for Clinicians 44, 43 (1994). Therapeutic agents that may be administered in combination with an antibody of the present invention as described elsewhere herein, such as, e.g., anti-cancer cytokines or chemokines, are also candidates for therapeutic moieties useful for conjugation to an antibody of the present invention.

In one embodiment, a polypeptide used in the present invention comprises a conjugated nucleic acid or nucleic acid-associated molecule. In one such embodiment, the conjugated nucleic acid is a cytotoxic ribonuclease, an antisense nucleic acid, an inhibitory RNA molecule (e.g., a siRNA molecule) or an immunostimulatory nucleic acid (e.g., an immunostimulatory CpG motif-containing DNA molecule). In another embodiment, a polypeptide used in the present invention is conjugated to an aptamer or a ribozyme.

In one embodiment, polypeptides comprising one or more radiolabeled amino acids are provided. Non-limiting examples of labels for polypeptides include ³ H, ¹⁴ C, ¹⁵ N, ³⁵ S, ⁹⁰ Y, "TC, ¹² 5I, ¹³¹ I, and ¹⁸⁶ Re. Methods for preparing radiolabeled amino acids and related peptide derivatives are known in the art, (see, for instance Junghans et a/., in Cancer Chemotherapy and Biotherapy 655-686 (2 ^nd Ed., Chafner and Longo, eds., Lippincott Raven (1996)) and U.S. 4,681,581, U.S. 4,735,210, U.S. 5,101,827, U.S. 5,102,990 (US RE35,500), U.S. 5,648,471 and U.S. 5,697,902. For example, a radioisotope may be conjugated by the chloramine-T method.

In one embodiment, a polypeptide or antibody used in the present invention is conjugated to a radioisotope or to a radioisotope-containing chelate. For example, the polypeptide can be conjugated to a chelator linker, e.g. DOTA, DTPA or tiuxetan, which allows for the polypeptide to be complexed with a radioisotope. Non-limiting examples of radioisotopes include ³ H, ¹⁴ C, ¹⁵ N, ³⁵ S, ⁹⁰ Y, "Tc, ¹²⁵ I, ⁱⁿ In, ¹³¹ I, ¹⁸⁶ Re, ²¹³ BS, ²²⁵ AC and ²²⁷ Th.

In one embodiment, a polypeptide or antibody used in the present invention may be conjugated to a cytokine selected from the group consisting of IL-2, IL-4, IL-6, IL-7, IL- 10, IL-12, IL-13, IL- 15, IL-18, IL-23, IL-24, IL-27, IL-28a, IL-28b, IL-29, KGF, IFNa, IFN , IFNy, GM-CSF, CD40L, Flt3 ligand, stem cell factor, ancestim, and TNFa.

Polypeptides or antibodies used in the present invention may also be chemically modified by covalent conjugation to a polymer to for instance increase their circulating half-life. Exemplary polymers, and methods to attach them to peptides, are illustrated in for instance US 4,766,106, US 4, 179,337, US 4,495,285 and US 4,609,546. Additional polymers include polyoxyethylated polyols and polyethylene glycol (PEG) (e.g. , a PEG with a molecular weight of between about 1,000 and about 40,000, such as between about 2,000 and about 20,000).

Conjugation to a therapeutic moiety may take place at the C-terminus of the polypeptide or at another site, typically at a site which does not interfere with oligomer formation.

Any method known in the art for conjugating the polypeptide or antibody used in the present invention to the conjugated molecule(s), such as those described above, may be employed, including the methods described by Hunter et a/. , Nature 144. 945 (1962), David et a/., Biochemistry 13, 1014 (1974), Pain et a/., J. Immunol. Meth. 4Ό, 219 ( 1981) and Nygren, J. Histochem. and Cytochem. 30, 407 ( 1982). Such variants may be produced by chemically conjugating the other moiety to the N-terminal side or C-terminal side of the variant or fragment thereof (e.g., an antibody H or L chain) (see, e.g., Antibody Engineering Handbook, edited by Osamu Kanemitsu, published by Chijin Shokan (1994)). Such conjugated variant derivatives may also be generated by conjugation at internal residues or sugars, where appropriate.

The agents may be coupled either directly or indirectly to a polypeptide or antibody used in the present invention. One example of indirect coupling of a second agent is coupling via a spacer or linker moiety to cysteine or lysine residues in an antibody. In one embodiment, a polypeptide or antibody is conjugated to a prodrug molecule that can be activated in vivo to a therapeutic drug. In some embodiments, the linker is cleavable under intracellular conditions, such that the cleavage of the linker releases the drug unit from the antibody in the intracellular environment. In some embodiments, the linker is cleavable by a cleavable agent that is present in the intracellular environment (e.g. within a lysosome or endosome or caveola). For example, the spacers or linkers may be cleavable by tumor-cell associated enzymes or other tumor-specific conditions, by which the active drug is formed. Examples of such prodrug technologies and linkers are described in W002083180, W02004043493, W02007018431, W02007089149, W02009017394 and

W0201062171 by Syntarga BV, et al. Suitable antibody-prodrug technology and duocarmycin analogs can also be found in U.S. Patent No. 6,989,452 (Medarex). The linker can also or alternatively be, e.g. a peptidyl linker that is cleaved by an intracellular peptidase or protease enzyme, including but not limited to, a lysosomal or endosomal protease. In some embodiments, the peptidyl linker is at least two amino acids long or at least three amino acids long. Cleaving agents can include cathepsins B and D and plasmin, all of which are known to hydrolyze dipeptide drug derivatives resulting in the release of active drug inside the target cells (see e. g. Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123). In a specific embodiment, the peptidyl linker cleavable by an intracellular protease is a Val-Cit (valine-citrulline) linker or a Phe-Lys (phenylalanine-lysine) linker (see e.g. US6214345, which describes the synthesis of doxorubicin with the Val-Cit linker and different examples of Phe-Lys linkers). Examples of the structures of a Val-Cit and a Phe-Lys linker include but are not limited to MC-vc-PAB described below, MC-vc- GABA, MC-Phe-Lys-PAB or MC-Phe-Lys-GABA, wherein MC is an abbreviation for maleimido caproyl, vc is an abbreviation for Val-Cit, PAB is an abbreviation for p- aminobenzylcarbamate and GABA is an abbreviation for g-aminobutyric acid.

Methods of preparing polypeptides of the invention, such as antibodies

Polypeptides of the invention, such as antibodies, are typically produced recombinantly, i.e. by expression of nucleic acid constructs encoding the polypeptides in suitable host cells, followed by purification of the produced recombinant polypeptide from the cell culture. Nucleic acid constructs can be produced by standard molecular biological techniques well-known in the art. The constructs are typically introduced into the host cell using a vector.

Suitable nucleic acid constructs, vectors are known in the art, and described in the Examples. In most embodiments, the polypeptide comprises not only a heavy chain (or Fc-containing fragment thereof) but also a light chain. In such embodiments, the nucleotide sequences encoding the heavy and light chain portions will typically be expressed in the same cells and may be present on the same or different nucleic acids or vectors.

Host cells suitable for the recombinant expression of antibodies are well- known in the art, and include CHO, HEK-293, Expi293F, PER-C6, NS/0 and Sp2/0 cells.

In one embodiment, said host cell is a cell which is capable of Asn-linked glycosylation of proteins, e.g. a eukaryotic cell, such as a mammalian cell, e.g . a human cell. In a further embodiment, said host cell is a non-human cell which is genetically engineered to produce glycoproteins having human-like or human glycosylation. Examples of such cells are genetically-modified Pichia pastoris (Hamilton et al., Science 301 (2003) 1244-1246; Potgieter et al., J . Biotechnology 139 (2009) 318-325) and genetically-modified Lemna minor (Cox et al., Nature Biotechnology 12 (2006) 1591-1597).

In one embodiment, said host cell is a mammalian or non-mammalian cell which produces homogenous glycoforms. In a further embodiment, said host cell is genetically engineered to produce glycoengineered antibodies, such as e.g. antibodies without core fucose. Examples of CHO cells producing defucosylated antibodies include Lecl3 cells and genetically-modified CHO cells, such as GDP- mannose-4, 6-dehydratase (GMD) knockout cells; GDP-fucose transporter knockout cells; FUT8 knockout cells; RNAi of FUT8 and/or GMD; or cells overexpressing GlcNAc transferase III or RMD (GDP-6-deoxy-d-lyxo-4-hexulose reductase) (reviewed in Li et al. 2017 Front Immunol 13;8 : 1554).

In one embodiment, said host cell is a host cell which is not capable of efficiently removing C-terminal lysine K447 residues from antibody heavy chains. For example, Table 2 in Liu et al. (2008) J Pharm Sci 97 : 2426 (incorporated herein by reference) lists a number of such antibody production systems, e.g . Sp2/0, NS/0 or transgenic mammary gland (goat), wherein only partial removal of C-terminal lysines is obtained.

The present invention is further illustrated by the following examples which should not be construed as further limiting. Table 1 SEQUENCE LIST

Table 2 self-oligomerization inhibiting substitutions*

Table 2* each column show self-oligomerization inhibiting substitutions, each row show Complementary self- self-oligomerization inhibiting Table 3 Substitutions that were tested in examples 5-23.

EXAMPLES

Example 1 : Antibody generation, production and purification

Expression constructs for antibodies

For the expression of human and humanized antibodies used herein, variable heavy (VH) chain and variable light (VL) chain sequences were prepared by gene synthesis (GeneArt Gene Synthesis; ThermoFisher Scientific, Germany) and cloned in pcDNA3.3 expression vectors (ThermoFisher Scientific, US) containing a constant region of a human IgG heavy chain (HC) (constant region human IgGlm(f) HC: SEQ ID NO 22; constant region human IgG2 HC: SEQ ID NO 31 ; constant region human IgG3 HC: SEQ ID NO 32; or constant region human IgG4 HC: SEQ ID NO 33) and or the constant region of the human kappa light chain (LC) : SEQ ID NO 34. Desired mutations were introduced by gene synthesis. CD20 antibody variants in this application have VH and VL sequences derived from previously described CD20 antibodies (W02004/035607) IgGl-CD20-7D8 (VH : SEQ ID NO 35; VL: SEQ ID NO 39) and IgGl-CD20-l lB8 (VH : SEQ ID NO 8; VL: SEQ ID NO 12). CD52 antibody variants in this application have VH and VL sequences derived from previously described CD52 antibody CAMPATH-1H (alemtuzumab; Crowe et al., 1992 Clin Exp Immunol. 87(1) : 105-110; VH : SEQ ID NO 1; VL: SEQ ID NO 5) CD37 antibody variants in this application have VH and VL sequences derived from previously described CD37 antibody IgGl-CD37-37.3 (WO2011/112978; VH : SEQ ID NO 42; VL: SEQ ID NO 46). DR5 antibody variants in this application have VH and VL sequences derived from previously described DR5 antibody DR5-01-G56T (WO 2017/093447; VH : SEQ ID NO 49; VL: SEQ ID NO 53) and DR5-05 (WO2014/009358; VH : SEQ ID NO 56; VL: SEQ ID NO 60). The human IgGl antibody bl2, an HIV gpl20-specific antibody was used as a negative control in some experiments (Barbas et al., J Mol Biol. 1993 Apr 5;230(3) :812-23; VH : SEQ ID NO 15; VL: SEQ ID NO 19).

Transient expression

Antibodies were expressed as IgGlK. Plasmid DNA mixtures encoding both heavy and light chains of antibodies were transiently transfected in Expi293F cells (Gibco, Cat # A14635) using 293fectin (Life Technologies) essentially as described by Vink et al. (Vink et al., Methods, 65 ( 1), 5-10 2014). Antibody concentrations in the supernatants were measured by absorbance at 280 nm. Antibodies were either directly used in in vitro assays, or purified as described below. Purification and analysis of proteins

Antibodies were purified by protein A affinity chromatography. Culture supernatants were filtered over a 0.20 mM dead-end filter and loaded on 5 ml_ MabSelect SuRe columns (GE Healthcare), washed and eluted with 0.02 M sodium citrate-NaOH, pH 3. The eluates were loaded on a HiPrep Desalting column (GE Healthcare) immediately after purification and the antibodies were buffer exchanged into 12.6 mM NaH ₂ P0 ₄ , 140 mM NaCI, pH 7.4 buffer (B. Braun or Thermo Fisher). After buffer exchange, samples were sterile filtered over 0.2 pm dead-end filters. Purified proteins were analyzed by a number of bioanalytical assays including capillary electrophoresis on sodium dodecyl sulfate-polyacrylamide gels (CE-SDS) and high- performance size exclusion chromatography (HP-SEC). Concentration was measured by absorbance at 280 nm. Purified antibodies were stored at 2-8°C.

Example 2: CDC activity of IgGl-Campath-E430G variants with mutations at positions 1253 or H310

We searched the Fc-Fc interface in the crystal structure of IgGl antibody bl2 (Protein Data Bank 1HZH; Oilman Sapphire et al, Science 2001, 293(5532): 1155- 1159) for intermolecular amino acid pairs that showed sterical proximity and side- chain orientations at opposite sides of the Fc-Fc interface. Mutation pairs were tested for complementarity in controlling intermolecular Fc-Fc interactions between cell- surface-target-bound antibodies, by interfering with the Fc-Fc interactions between the antibodies that have the same mutation, and rescuing Fc-Fc interactions by mixtures of two antibodies, each harboring one and the other mutation. The amino acid pair 1253 + H310 was selected for extensive mutagenesis and functional characterization. An antibody mutant library was generated based on the positions 1253 and H310 by substituting isoleucine at position 253 and histidine at position 310 by any other amino acid except cysteine or proline, and introducing the mutations in IgGl-Campath-E430G (i.e. antibody Campath-1H containing heavy chain constant domain SEQ ID: 26, comprising the Fc-Fc interaction-enhancing Glu to Gly mutation at position 430 (W02013004842)). The effects of the individual mutations on positions 253 and 310 and of all possible 1253 and H310 mutation pairs on the CDC efficacy of the respective IgGl-Campath-E430G variants and mixtures thereof were subsequently tested in an in vitro CDC assay using Wien 133 cells (kindly provided by Dr. Geoff Hale, BioAnaLab Limited, Oxford, UK). Cells were harvested and resuspended in medium [RPMI (Lonza, Cat # BE12-115F) with 0.2% bovine serum albumin (BSA; Roche Cat # 10735086001)]. 5,000 cells per well were incubated with concentration series of the single antibodies and antibody combinations ( 15.6-2000 ng/ml_ final antibody concentrations in 2-fold dilutions; dilutions of supernatants of transient transfections as described in Example 1) in the presence of 5% normal human serum (NHS; Sanquin, Ref # M0008) as a source of human complement. Simultaneously, TO-PRO-3 iodide (ThermoFischer Scientific, CAT # T3605, 1 mM final concentration) was added as a cell viability marker and SYBR Green I (ThermoFischer Scientific, Cat # S7563; 12,500 x diluted from original stock concentrate) was added to detect the presence of cells. Assay plates were incubated for one hour at room temperature and killing was calculated as the fraction of TO- PRO-3 iodide-positive cells (%) as determined by flow cytometry using a Celigo Imaging Cytometer (Brooks Life Science Systems).

Introduction of several tested 1253 and H310 amino acid substitutions resulted in inhibition of CDC efficacy of IgGl-Campath-E430G, as represented by an increased EC50 value (summarized in Figure 2; EC50 value of IgGl-Campath-E430G was < 15 ng/pL). Most mixtures of IgGl-Campath-E430G variants each containing a mutation at either position 253 or 310, did not overcome the inhibition of CDC efficacy mediated by the single antibodies. However, an exception was the mutation pair I253G (Ile253 -> Gly) + H310R (His310 -> Arg), which each showed CDC inhibition when introduced and tested as single IgGl-Campath-E430G variants (containing the I253G or H310R mutation), but complete rescue of CDC efficacy when tested as a mixture of the two IgGl-Campath-E430G variants, each containing either I253G or H310R. Furthermore, for the mutation pairs I253K (Ile253 -> Lys) + H310D (His310 -> Asp), and I253R (Ile253 -> Arg) + H310D, CDC inhibition was observed for the single IgGl-Campath-E430G variants (containing the I253K, I253R or H310D mutation), and partial rescue of CDC efficacy by the mixture of the two IgGl- Campath-E430G variants with one containing the I253K or I253R mutation and the other H310D (Figure 2).

Based on these results, it can be concluded that it is unpredictable which Fc mutations at positions 1253 and H310 in human IgGl antibodies with an E430G Fc- Fc-enhancing mutation create complementary mutation pairs that show inhibition of Fc-Fc interactions and inhibition of CDC efficacy by the single variants containing either a 1253 or a H310 mutation, and rescue thereof by mixing the two variants, each containing one of the two complementary 1253 and H310 mutations. Using the CDC assay with IgGl-Campath-E430G antibody variants on positions 1253 and H310 on Wien 133 cells, I253G + H310R, I253K + H310D and I253R + H310D were identified as complementary mutation pairs that showed control of CDC activity of the antibody with the E430G Fc-Fc-enhancing mutation, i.e. inhibition of CDC efficacy by the single variants (I253G, I253K, H310D or H310R) and rescue by the complementary mixtures (I253G + H310R, I253K + H310D or I253R + H310D) thereof.

Example 3: CDC activity of IgGl-Campath-E430G variants with mutations at positions Y436 or Q438

Similar to the amino acid pair 1253 + H310 described in Example 2, also the amino acid pair Y436 + Q438 was selected for extensive mutagenesis and functional characterization. An antibody mutant library was generated based on the positions Y436 and Q438 by substituting tyrosine at position 436 and glutamine at position 438 by any other amino acid except cysteine or proline in IgGl-Campath-E430G. The effects of the individual mutations on positions 436 and 438 and of all possible Y436 and Q438 mutation pairs on the CDC efficacy of the respective IgGl-Campath-E430G variants and mixtures thereof were subsequently tested in an in vitro CDC assay using Wien 133 cells as described in Example 2.

Introduction of several tested Y436 and Q438 amino acid substitutions resulted in inhibition of CDC efficacy of IgGl-Campath-E430G, as represented by an increased EC50 value (summarized in Figure 3; EC50 value of IgGl-Campath-E430G was < 15 ng/ml_). Many mixtures of IgGl-Campath-E430G variants each containing a mutation at either position 436 or 438 did not overcome the inhibition of CDC efficacy mediated by the single antibodies. However, partial rescue of CDC efficacy was observed for mixtures of IgGl-Campath-E430G variants that brought together the mutation pairs Y436K + Q438G, Y436K + Q438H, Y436K + Q438K, Y436K + Q438N, Y436K + Q438R, Y436N + Q438G, Y436N + Q438H, Y436N + Q438K, Y436N + Q438N, Y436N + Q438R, Y436Q + Q438G, Y436Q + Q438H, Y436Q + Q438K, Y436Q + Q438N, Y436Q + Q438R, Y436R + Q438G, Y436R + Q438H, Y436R + Q438K, Y436R + Q438N, or Y436R + Q438R (Figure 3).

Based on these results, it can be concluded that it is unpredictable which Fc mutations at positions Y436 and Q438 in human IgGl antibodies with an E430G Fc- Fc-enhancing mutation will show inhibition of Fc-Fc interactions by the single mutants and whether a specific mixture of two variants could rescue it. Using the CDC assay with IgGl-Campath-E430G antibody variants on positions Y436 and Q438 on Wien 133 cells, the Y436K, Y436N, Y436Q, Y436R, Q438G, Q438H, Q438K, Q438N and Q438R mutations were identified that can inhibit Fc-Fc interactions and CDC activity of the antibody with the E430G Fc-Fc-enhancing mutation, and any mixture of one of these mutations at position 436 with one of these mutations at position 438 was identified as a complementary Y436;Q438 mutation pair that can rescue the inhibition of Fc-Fc interactions and CDC efficacy of the single mutants.

Example 4: CDC activity of IgGl-Campath-E430G variants with mutations at positions K439 or S440

Similar to the amino acid pairs 1253 + H310 (described in Example 2) and Y436 + Q438 (described in Example 3), also the amino-acid pair K439 + S440 was selected for extensive mutagenesis and functional characterization. An antibody mutant library was generated based on the positions K439 and S440 by substituting lysine at position 439 and serine at position 440 by any other amino acid except cysteine or proline in IgGl-Campath-E430G. The effects of the individual mutations on positions 439 and 440 and of all possible K439 and S440 mutation pairs on the CDC efficacy of the respective IgGl-Campath-E430G variants and mixtures thereof were subsequently tested in an in vitro CDC assay using Wien 133 cells as described in Example 2.

Introduction of several tested K439 and S440 amino acid substitutions resulted in inhibition of CDC efficacy of IgGl-Campath-E430G, as represented by an increased EC50 value (summarized in Figure 4; EC50 value of IgGl-Campath-E430G was < 15 ng/ml_). Many mixtures of IgGl-Campath-E430G variants, each containing a mutation at either position 439 or 440, did not overcome the inhibition of CDC efficacy mediated by the single antibodies. Only for the mutation pairs in which S440K was combined with K439E, K439F K439I, K439Y, K439T, K439V or K439W, CDC inhibition was observed when introduced and tested as single IgGl-Campath- E430G variants, and rescue of CDC efficacy when tested as a mixture of the two IgGl-Campath-E430G variants, each containing either the S440K mutation or one of these K439 mutations (Figure 4).

Based on these results, it can be concluded that it is unpredictable which Fc mutations at positions K439 and S440 in human IgGl antibodies with an E430G Fc- Fc-enhancing mutation create complementary mutation pairs that show inhibition of Fc-Fc interactions and inhibition of CDC efficacy by the single variants containing one or the other K439 or S440 mutation, and rescue thereof by the mixture of the two, each containing one of the two complementary K439 and S440 mutations. Using the CDC assay with IgGl-Campath-E430G antibody variants on positions K439 and S440 on Wien 133 cells, K439E + S440K, K439F + S440K, K439I + S440K, K439Y + S440K, K439T + S440K, K439V + S440K and K439W + S440K were identified as complementary mutation pairs that showed control of CDC activity of the antibody with the E430G Fc-Fc-enhancing mutation, i.e. inhibition of CDC efficacy by the single variants and rescue by the complementary mixtures thereof.

Example 5: Validation of complementary mutation pairs at positions 1253 and H310 of a human IgGl-E430G antibody

The control of Fc-Fc interactions and CDC efficacy by the complementary 1253 + H310 mutation pairs, identified in the CDC assay described in Example 2, was validated in in vitro CDC assays with further concentration titration series of purified antibodies. 0.1 x 10 ⁶ Wien 133 cells were pre-incubated in polystyrene round-bottom 96-well plates (Greiner bio-one Cat # 650101) with concentration series of purified samples of IgGl-Campath-E430G variants (final concentration range 0.03 - 10.0 pg/mL in 3-fold dilution steps) in 80 pl_ culture medium (RPMI with 0.2% BSA) for 15 min on a shaker at room temperature. Next, 20 mI_ NHS was added as a source of complement (20% final NHS concentration) and incubated for 45 minutes at 37°C. The reaction was stopped by putting the plates on ice before pelleting the cells by centrifugation and replacing the supernatant by 30 mI_ of 1.67 pg/mL propidium iodide solution (PI; Sigma Aldrich, Zwijnaarde, The Netherlands). CDC efficacy was determined by the percentage Pi-positive cells measured by flow cytometry using an Intellicyt iQue™ screener (Westburg). The data were analyzed using best-fit values of a non-linear dose-response fit using log-transformed concentrations in GraphPad PRISM 7.02 (GraphPad Software, San Diego, CA, USA). The percentage lysis was calculated as (number of Pi-positive cells / total number of cells) x 100%.

Introduction of I253G or H310R resulted in CDC inhibition for the single IgGl- Campath-E430G antibody variants containing either the I253G or H310R mutation, and complete rescue of CDC efficacy in the mixture thereof (Figure 5A).

Introduction of I253K, I253R or H310D resulted in CDC inhibition for the single IgGl- Campath-E430G antibody variants containing either the I253K, I253R or H310D mutation, and partial rescue of CDC efficacy by the mixtures of two antibody variants that brought together the mutation pairs I253K + H310D (Figure 5B) or I253R + H310D (Figure 5C). Together, these results confirmed that the introduction of the complementary mutation pairs I253G + H310R, I253K + H310D, or I253R + H310D identified in Example 2, can be used to control Fc-Fc interactions and CDC efficacy in mixtures of two human IgGl antibodies with an Fc-Fc-enhancing background mutation, such as E430G.

Example 1: Combinations of complementary Fc-Fc inhibiting mutation pairs in human IgGl

With the aim to further suppress the CDC activity of single antibody-agents while retaining high potency of the mixed antibody pairs, some of the Fc mutations that resulted in efficient inhibition of CDC efficacy in the CDC assays described in Example 2 and Example 3 were combined with the self-oligomerization inhibiting mutations K439E or S440K (Example 4; W02013004842) in IgGl-Campath-E430G. The effect of these mutation combinations was tested in an in vitro CDC assay using Wien 133 cells as described in Example 5. The complementary mutation pairs I253G + H310R, I253K + H310D, I253R + H310D (identified in Example 2), Y436N + Q438R, and Q438N + Y436K (identified in Example 3) were pairwise combined with K439E and S440K, resulting in the Fc-Fc self-oligomerization inhibition double mutant pairs I253G/K439E + H310R/S440K, H310D/K439E + I253K/S440K, H310D/K439E + I253R/S440K, Y436N/K439E + Q438R/S440K, and Q438N/K439E + Y436K/S440K. Furthermore, also the following combinations of K439E + S440K with different Y436 or Q438 mutations (identified in Example 3) were tested in IgGl-Campath-E430G: Y436N/K439E (containing SEQ ID NO:81) + Y436K/S440K (containing SEQ ID NO: 80) and Q438N/K439E (containing SEQ ID NO: 74)+ Q438R/S440K (containing SEQ ID NO: 77).

Introduction of all tested self-oligomerization inhibiting double mutations resulted in stronger inhibition of CDC than the IgGl-Campath-E430G antibody variants with only one self-oligomerization inhibiting mutation, K439E or S440K (Figure 6). For the tested combinations of mutation pairs, complete rescue of CDC efficacy was observed for the I253G/K439E + H310R/S440K (Figure 6A), H310D/K439E + I253K/S440K (Figure 6B), H310D/K439E + I253R/S440K (Figure 6C) and the Y436N/K439E + Q438R/S440K mutation pairs (Figure 6D), which are thus complementary mutation pairs showing a large window to control CDC efficacy (difference between inhibited and rescued CDC efficacy of the single antibodies and combination thereof, respectively). Partial recovery of CDC activity was observed for the mixture of IgGl-Campath- E430G antibody variants with the mixture of Q438N/K439E and Y436K/S440K (Figure 6E) and, unexpectedly because not identified in previous examples, also the mixture Q438N/K439E + Q438R/S440K (Figure 6F). These mixtures showed higher CDC efficacy than the individual antibodies, but lower CDC efficacy than the parental antibody IgGl-Campath-E430G without a self-oligomerization inhibiting mutation or the antibody combination of two IgGl-Campath-E430G variants with only one self- oligomerization inhibiting mutation pair, K439E + S440K (Figure 6E/F).

No rescue of CDC efficacy was observed for the combination of IgGl-Campath- E430G antibody variants with the Fc-Fc inhibiting double mutation pair Y436N/K439E + Y436K/S440K (Figure 6G).

Taken together, it was shown for antibodies with an Fc-Fc interaction enhancing mutation, such as E430G, that the "window" between inhibited and rescued CDC efficacy by self-oligomerization inhibiting mutation pairs can be tuned by combining more than one self-oligomerization inhibiting mutation in each antibody, although the breadth of the window that was reached by the different combinations of IgGl- Campath-E430G variants was unpredictable.

Example 7: FcRn binding of anti-CD52 IgG 1-CAM PATH- 1H antibody variants

The neonatal Fc receptor (FcRn) is responsible for the long plasma half-life of IgG by protecting IgG from degradation. After internalization of the antibody, FcRn binds to antibody Fc regions in endosomes, where the interaction is stable in the mildly acidic environment (pH 6.0). Upon recycling to the plasma membrane, where the environment is neutral (pH 7.4), the interaction is lost and the antibody is released back into the circulation. This influences the plasma half-life of IgG.

An FcRn binding enzyme-linked immunosorbent assay (ELISA) was performed to evaluate binding of human FcRn to anti-CD52 IgGl-CAMPATH-lH containing the hexamerization enhancing mutation E430G, one of the self-oligomerization inhibiting mutations K439E or S440K, and one of the self-oligomerization inhibiting mutations I253G, I253K, I253R, H310D, or H310R identified in Example 2, or Y436K, Y436N, Q438N, or Q438R identified in Example 3. Streptawell 96 well plates (Roche, Cat No. 1734776001) were coated with 5 pg/mL (100 pL/well) recombinantly produced biotinylated extracellular domain of human FcRn [FcRnECDHis-B2M-BIO, i.e. the extracellular domain of human FcRn with a C-terminal His tag (FcRnECDHis; SEQ ID NO: 111) as dimer with beta2microglobulin (B2M; SEQ ID NO: 112), diluted in PBS supplemented with 0.05% Tween 20 (PBST) plus 0.2% BSA for 2 hours while shaking at room temperature (RT). Plates were washed three times with PBST. Serially diluted antibody samples (range 0.003-40 pg/mL final concentrations in 5- fold dilutions in PBST/0.2% BSA, pH 6.0 or pH 7.4) were added and incubated for 1 hour at RT while shaking . Plates were washed with PBST/0.2% BSA, pH 6.0 or pH 7.4. Horseradish Peroxidase (HRP)-conjugated polyclonal Goat-anti-Human kappa light chain (1 : 5,000; Sigma, Cat No. A-7164) diluted in PBST/0.2% BSA, pH 6.0 or pH 7.4 was added, and plates were incubated for 1 hour at RT while shaking . After washing with PBST/0.2% BSA, pH 6.0 or pH 7.4., 100 pl_ 2,2'-Azino-bis(3- ethylbenzthiazoline-6-sulfonic acid (ABTS; 1 mg/ml_; Roche Cat No. 11112422001 and 11112597001) was added as substrate and plates were incubated for 10 minutes at RT protected from light. The reaction was stopped using 100 pl_ 2% oxalic acid (Riedel de Haen, Cat No. 33506), incubated for 10 minutes at RT and absorbance was measured at 405 nm using an ELISA reader. Log-transformed data were analyzed by fitting sigmoidal dose-response curves with variable slope using GraphPad Prism software.

All tested IgGl-CAMPATH-lH antibody variants showed no binding to human FcRn at pH 7.4. At pH 6.0, antibodies IgGl-bl2, wild-type anti-CD52 IgGl-CAMPATH-lH and anti-CD52 IgGl-CAMPATH- lH variants E430G, K439E did show binding, as well as the antibody variants of anti-CD52 IgGl-CAMPATH- lH with mutations at amino acid position Y436 and Q438 (Figure 7). In addition, the S440K mutation did not inhibit FcRn binding. In contrast, no binding to FcRn at pH 6.0 was observed by antibody variants of anti-CD52 IgGl-CAMPATH-lH with mutations at amino acid position 1253 and H310. Taken together, these results show that anti-CD52 IgG 1-CAM PATH- 1H with hexamerization enhancing mutation E430G and self-oligomerization inhibiting mutations K439E, S440K, Y436K, Y436N, Q438N and/or Q438R showed normal binding to human FcRn, while the ability to bind FcRn was lost by introduction of self- oligomerization inhibiting mutations H310D, H310R, I253G, I253K or I253R.

Example 8: The effect of Y436K, Y436N, Q438N and Q438R mutations on the in vitro FcyR binding of anti-CD52 antibodies with a hexamerization enhancing mutation and K439E or S440K

Using purified antibodies, binding of IgGl-CAMPATH-lH to dimeric ECDs of FcyRIIA allotype 131H (SEQ ID NO: 113), FcyRIIA allotype 131R (SEQ ID NO: 114), FcyRIIB (SEQ ID NO: 115), FcyRIIIA allotype 158F (SEQ ID NO: 116), and FcyRIIIA allotype 158V (SEQ ID NO : 117) was tested in ELISA assays. To detect binding to dimeric FcyR variants, 96-well Microlon ELISA plates (Greiner, Germany) were coated overnight at 4 °C with goat F(ab')2-anti-human-IgG-F(ab')2 (Jackson Laboratory, 109-006-097, 1 gg/mL) in PBS, washed and blocked with 200 pL/well PBS/0.2% BSA for 1 h at room temperature (RT). With washings in between incubations, plates were sequentially incubated with 100 pL/well of a dilution series of IgGl-CAMPATH- 1H antibody variants (0.0013-20 gg/mL in five-fold steps) in PBST/0.2% BSA for 1 h at RT while shaking, 100 pL/well of dimeric, His-tagged, C-terminally biotinylated FcyR ECD variants (1 gg/mL) in PBST/0.2% BSA for 1 h at RT while shaking, and with 100 pL/well Streptavidin-polyHRP (CLB, M2032, 1 : 10.000) in PBST/0.2% BSA as detecting antibody for 30 min at RT while shaking. Development was performed for circa 24 (IIB) or 30 (IIA-131H, IIA-131R, IIIA-158V, IIIA-158F) min with 1 mg/mL ABTS (Roche, Mannheim, Germany). To stop the reactions, 100 pL/well of 2% oxalic acid was added. Absorbances were measured at 405 nm in a microplate reader (BioTek, Winooski, VT). FcyR binding at 20 gg/mL antibody concentration was plotted. Data is based on three independent replicates, normalized per experiment relative to background signal in ELISA (no antibody control, 0%) and an internal standard, IgGl-CAMPATH-lH-E430G, set to 100%.

Certain applications of co-dependent antibody mixtures with regulated Fc-Fc interaction properties may require the presence of intact FcyR-mediated effector functions. Assessment of binding of IgGl-CAMPATH-lH variants with Fc-Fc interaction enhancing mutation E430G and self-oligomerization inhibiting mutations K439E, S440K, Y436K, Y436N, Q438N and Q438R to FcyRIIa, FcyRIIb and FcyRIIIa by ELISA revealed that all tested antibodies retained FcyR binding at an antibody concentration of 20 pg/ml (Figure 8A-E). A relatively lower FcyR-binding was observed for variants IgGl-CAMPATH-lH-E430G-S440K-Y436K and IgGl-CAMPATH- 1H-E430G-S440K-Q438R.

In conclusion, IgGl-CAMPATH-lH antibody variants with Fc-Fc interaction enhancing mutation E430G and self-oligomerization inhibiting mutations K439E, S440K, Y436K, Y436N, Q438N and Q438R retained FcyR binding.

Example 9: Selectivity of CDC activity by mixed antibody variants by introduction of Fc-Fc self-oligomerization inhibiting mutations in anti-CD52 IgGl-CAMPATH-lH with an E430G Fc-Fc interaction enhancing mutation

The effect of self-oligomerization inhibiting mutations Y436K, Y436N, Q438N, Q438R, K439E, and S440K on in vitro CDC efficacy was tested using mixtures of variants of anti-CD52 antibody IgGl-CAMPATH-lH with an E430G Fc-Fc interaction enhancing mutation. Different concentrations of purified antibodies (range 0.01-40.0 pg/mL final concentrations) were tested in an in vitro CDC assay on Wien 133 cells with 20% NHS. Different mutations were introduced in antibody IgGl-CAMPATH-lH : E430G, which induces enhanced Fc-Fc interactions; and one or more of the self- oligomerization inhibiting mutations Y436K, Y436N, Q438N, Q438R, K439E, or S440K. As controls, single antibodies were also mixed 1 : 1 with non-binding isotype control antibody IgGl-bl2 to enable direct comparison of the concentrations of individual components and mixtures composed thereof; these conditions will be referred to as single agent activity hereafter. For the CDC assay, 0.1 x 10 ⁶ Wien 133 cells (kindly provided by Dr. Geoff Hale, BioAnaLab Limited, Oxford, UK) in RPMI (Sigma, Cat No. R5886-500mL) with 0.2% bovine serum albumin (BSA; Roche, Cat No. 10735086001) were pre-incubated in polystyrene round-bottom 96-well plates (Greiner bio-one Cat No. 650180) with a concentration series of purified antibodies in a total volume of 80 pL for 15 min on a shaker at RT. Next, 20 pL normal human serum (NHS; Sanquin) was added as a source of complement and the mixture was incubated in a 37°C incubator for 45 min (20% final NHS concentration; 40 to 0.01 pg/mL final IgG concentration in 3.3-fold dilutions). The reaction was stopped by putting the plates on ice before pelleting the cells by centrifugation and replacing the supernatant by 30 pL of 2 pg/mL propidium iodide solution (PI; Sigma Aldrich, Cat No. 1002570846). The number of Pi-positive cells was determined by flow cytometry on an Intellicyt iQue screener (Westburg) and the percentage of lysis was calculated as (number of Pi-positive cells / total number of cells) x 100%. The area under the dose-response curves with log-transformed concentrations of two experimental replicates was calculated and averaged using GraphPad Prism 7. Relative areas under the curve (AUC) values represent values normalized relative to lysis induced by non- binding control IgGl-bl2 (0%) and maximal lysis by anti-CD52 IgG 1-CAM PATH- 1H- E430G (100%).

Anti-CD52 antibody IgGl-CAMPATH-lH-E430G induced efficient lysis of Wien 133 cells (represented as Area Under the Curve (AUC); Figure 9A; set to 100%) compared to non-binding control IgG-bl2 (set to 0%). All other antibody samples contained total IgG concentrations equal to these control reactions, but were composed of two different antibodies mixed at 1: 1 ratio. Introduction of the K439E or S440K mutation in IgGl-CAMPATH-lH-E430G creating variants E430G-K439E and E430G-S440K resulted in decreased CDC efficacy when tested as a single agent in combination with IgGl-bl2 (Figure 9A), but both variants retained substantial single agent activity, particularly at 40 pg/mL IgG concentration (Figure 9B). When IgGl- CAM PATH- 1H-E430G-K439E and IgGl-CAMPATH-lH-E430G-S440K were mixed, CDC efficacy was recovered. The residual CDC efficacy of IgGl-Campath-lH-E430G- S440K when tested as single agent in combination with IgGl-bl2 was strongly decreased by introduction of either of the mutations Y436K, Y436N, Q438N or Q438R, including at 40 pg/mL IgG concentration (Figure 9B) . A strong decrease in CDC efficacy was also observed when either of the mutations Y436K, Y436N or Q438N was introduced in IgGl-CAMPATH-lH-E430G-K439E when tested as a single agent with IgGl-bl2. In stark contrast, introduction of mutation Q438R in IgGl- CAMPATH-1H-E430G-K439E increased CDC efficacy as a single agent in combination with IgGl-bl2 (Figure 9A, 9B).

When variants of IgGl-CAMPATH-lH-E430G-K439E and IgGl-CAMPATH-lH-E430G- S440K with self-oligomerization inhibiting mutations Y436K, Y436N, Q438N or Q438R were combined, substantial recovery of CDC efficacy (represented as AUC; Figure 9A) and maximal percentage of cell lysis (Figure 9B) was observed for all combinations, except when one antibody harboring the Y436K mutation and one antibody harboring the Y436N mutation were combined. The latter occurred both when combining IgGl-CAMPATH-lH-E430G-K439E-Y436N with IgG 1-CAM PATH- 1H- E430G-S440K-Y436K and when combining IgGl-CAMPATH-lH-E430G-K439E-Y436K with IgGl-CAMPATH-lH-E430G-S440K-Y436N. Collectively, these data demonstrate that the introduction of self-oligomerization inhibiting mutations Y436K, Y436N, Q438N or Q438R in IgGl-CAMPATH-lH-E430G-S440K and Y436K, Y436N and Q438N mutations in IgGl-CAMPATH-lH-E430G-K439E results in a further reduction of CDC efficacy of the single agents. CDC efficacy was recovered by mixing complementary IgG 1-CAM PATH- 1H-E430G-K493E and IgGl-CAMPATH- lH-E430G-S440K variants harboring self-oligomerization inhibiting mutations, with the exception of combinations of IgGl-CAMPATH-lH-E430G-K439E-Y436N with IgG 1-CAM PATH- 1H- E430G-S440K-Y436K and IgGl-CAMPATH-lH-E430G-K439E-Y436K with IgGl- CAMPATH-1H-E430G-S440K-Y436N.

Example 10: Selectivity of CDC activity by mixed antibody variants by introduction of Fc-Fc self-oligomerization inhibiting mutations in anti-CD20 IgGl-llB8 with an E430G Fc-Fc interaction enhancing mutation

The effect of self-oligomerization inhibiting mutations Y436K, Y436N, Q438N, Q438R, K439E, or S440K on in vitro CDC efficacy was tested using mixtures of variants of anti-CD20 antibody IgGl- 11B8 with an E430G Fc-Fc interaction enhancing mutation essentially as described in Example 9. Here, different mutations were introduced in CD20-directed antibody lgGl-llB8 instead: E430G, which induces enhanced Fc-Fc interactions; and one or more of the self-oligomerization inhibitory mutations Y436K, Y436N, Q438N, Q438R, K439E, or S440K. The area under the dose- response curves (AUC) with log-transformed concentrations of two experimental replicates was calculated using GraphPad Prism 7. The AUC was normalized per plate relative to lysis induced by non-binding control lgGl-bl2 (0%) and maximal lysis by the mixture of anti-CD52 IgGl- CAMPATH-1H-E430G + anti-CD20 lgGl-llB8-E430G (100%), and subsequently averaged over multiple experiments.

A mixture of anti-CD52 lgGl-CAMPATH-lH-E430G and anti-CD20 lgGl-HB8-E430G induced efficient lysis of Wien 133 cells (represented as Area Under the Curve (AUC); Figure 10; set to 100%) compared to non-binding control lgG-bl2 (set to 0%). Likewise, lgGl-llB8-E430G induced lysis of Wien 133 cells. No single agent activity was observed by the lgGl-llB8-E430G- S440K variants with self-oligomerization inhibiting mutations Y436K or Q438R, nor by the IgGl- 11B8-E430G-K439E variants with self-oligomerization inhibiting mutations Y436N or Q438N. By mixing lgGl-llB8-E430G-K439E-Y436N and lgGl-HB8-E430G-S440K-Q438R, CDC efficacy could be recovered to a level similar to that induced by lgGl-llB8-E430G. However, no recovery was observed for mixtures of lgGl-HB8-E430G-K439E-Y436N + lgGl-HB8-E430G-S440K-Y436K or lgGl-llB8-E430G-K439E-Q438N + lgGl-llB8-E430G-S440K-Y436K, while low CDC efficacy was recovered after mixing lgGl-llB8-E430G-K439E-Q438N + lgGl-llB8-E430G-S440K-Q438R (Figure 10).

Taken together, these results demonstrate that introduction of self-oligomerization inhibiting mutations Y436K, Y436N, Q438N or Q438R in lgGl-llB8-E430G that contain either of the self oligomerization inhibiting mutations K439E or S440K results in loss of single agent CDC efficacy in the Wien 133 cell in vitro CDC model. In this model of antibody-mediated CD20-targeting, CDC efficacy could only be recovered by mixing lgGl-llB8-E430G-K439E-Y436N and lgGl-llB8- E430G-S440K-Q438R.

Example 11: Selectivity of CDC activity by mixed antibody variants by introduction of self-oligomerization inhibiting mutations in anti-CD52 IgGl- CAMPATH-1H and anti-CD20 IgGl-llB8 with an E430G Fc-Fc interaction enhancing mutation

It was described in Examples 9 and 10 that introduction of self-oligomerization inhibiting mutations in either IgG 1-CAM PATH- 1H (Example 9) or IgGl-llB8 (Example 10) with the Fc-Fc interaction enhancing mutation E430G and either of the self-oligomerization inhibiting mutations K439E or S440K resulted in reduced single agent activity in an in vitro CDC model, while recovery of CDC efficacy was observed when mixing complementary antibody variants with said mutations targeting the same antigen. Here, the effect of introducing self-oligomerization inhibiting mutations in two antibodies targeting different antigens was tested, namely anti- CD20 IgGl-llB8 and anti-CD52 IgGl-CAMPATH-lH. CDC activity was tested essentially as described in Example 9. Different mutations were introduced in antibodies IgGl-llB8 and IgG 1-CAM PATH- 1H : E430G, which induces enhanced Fc- Fc interactions; and one or more of the self-oligomerization inhibiting mutations Y436K, Y436N, Q438N, Q438R, K439E, or S440K, which inhibit the formation of homo-hexameric antibody complexes and promote the formation of hetero- hexameric antibody complexes. The area under the dose-response curves (AUC) with log-transformed concentrations of two experimental replicates was calculated using GraphPad Prism 7. The AUC was normalized per plate relative to lysis induced by non-binding control IgGl-bl2 (0%) and maximal lysis by the mixture of anti-CD52 IgG 1-CAM PATH- 1H-E430G + anti-CD20 IgGl-llB8-E430G (100%), and subsequently averaged over multiple experiments.

A mixture of anti-CD52 IgGl-CAMPATH-lH-E430G and anti-CD20 IgGl-llB8-E430G induced efficient lysis of Wien 133 cells (represented as Area Under the Curve (AUC); Figure 11; set to 100%) compared to non-binding control IgG-bl2 (set to 0%). While CDC efficacy was fully abrogated by the introduction of mutation S440K in IgGl-llB8-E430G, introduction of mutation K439E in IgGl-CAMPATH-lH-E430G only reduced the single agent activity. CDC efficacy was recovered by mixing of antibodies IgG 1-CAM PATH- 1H-E430G-K439E and IgGl-llB8-E430G-S440K, hereafter referred to as the prior art mixture.

Introduction of the self-oligomerization inhibiting mutations Y436K, Y436N, Q438N or Q438R in IgGl-llB8-E430G-S440K resulted in complete loss of single agent CDC activity. Similar to the data presented in Example 9, introduction of mutations Y436K, Y436N or Q436N, but not Q438R, in IgGl-CAMPATH-lH-E430G-K439E resulted in low single agent activity as compared with IgGl-CAMPATH-lH-E430G- K439E.

Approximately 80 to 90% of the CDC efficacy induced by the prior art mixture could be restored by a mixture of IgGl-CAMPATH-lH-E430G-K439E-Y436K with IgGl- 11B8-E430G-S440K-Q438N or with IgGl-llB8-E430G-S440K-Q438R (but not IgGl- 11B8-E430G-S440K-Y438N). Approximately 80 to 90% of the CDC efficacy induced by the prior art mixture was also recovered by a mixture of IgGl-CAMPATH-lH- E430G-K439E-Y436N with IgGl-llB8-E430G-S440K-Q438N or with IgGl-llB8- E430G-S440K-Q438R (but not IgGl-llB8-E430G-S440K-Y436K). Mixtures of IgGl- CAMPATH-1H-E430G-K439E-Q438N with IgGl-llB8-E430G-S440K-Y436K, IgGl- 11B8-E430G-S440K-Y436N or IgGl-llB8-E430G-S440K-Q438R recovered up to 84% of the efficacy of the prior art mixture. Although single agent activity of IgGl- CAMPATH-1H-E430G-K439E-Q438R was relatively high, CDC efficacy was slightly further increased by mixing IgGl-CAMPATH-lH-E430G-K439E-Q438R with IgGl- 11B8-E430G-S440K-Y436K or IgGl-llB8-E430G-S440K-Y436N.

Collectively, these data demonstrate that the introduction of self-oligomerization inhibiting mutations Y436K, Y436N, Q438N or Q438R in IgGl-llB8-E430G-S440K and Y436K, Y436N and Q438N mutations in IgGl-CAMPATH-lH-E430G-K439E results in a further reduction of CDC efficacy of the single agents, confirming results described in Example 9 and 10. CDC efficacy was restored after mixing complementary antibodies targeting different antigens, demonstrated by the observation that CDC efficacy was restored by mixtures of IgGl-CAMPATH-lH- E430G-K493E and IgGl-llB8-E430G-S440K variants harboring said self- oligomerization inhibiting mutations, with the exception of combinations of IgGl- CAMPATH-1H-E430G-K439E-Y436N with IgGl-llB8-E430G-S440K-Y436K and IgGl- CAMPATH-1H-E430G-K439E-Y436K with IgGl-llB8-E430G-S440K-Y436N.

Example 12: Selectivity of CDC activity by mixed antibody variants of anti- CD52 IgGl-CAMPATH-lH + anti-CD20 IgGl-llB8 with different Fc-Fc interaction enhancing mutations

It was shown in Example 11 that the introduction of self-oligomerization inhibiting mutations could enhance CDC selectivity of two antibodies targeting different antigens. Here, we test the selectivity of CDC activity of antibody variants with different Fc-Fc interaction enhancing mutations, E430G, E345K, E345R and K248E/T437R.

An in vitro CDC assay using Wien 133 cells was performed with 20% NHS and antibody concentration series (final concentration range 0.01-40.0 pg/mL in 3.3-fold dilutions), essentially as described in Example 9. Cell lysis and relative AUC values were calculated from the number of Pi-positive cells as described in Example 9, from two experimental replicates. AUC was normalized to the values for negative control antibody IgGl-bl2 (0%) and for positive control IgGl-CAMPATH-lH-E430G + IgGl- 11B8-E430G (100%).

No cell lysis was observed by IgGl-bl2 (Figure 12A, B; set to 0%), while a 1: 1 mixture of anti-CD52 IgGl-CAMPATH-lH-E430G + anti-CD20 IgGl-llB8-E430G induced efficient cell lysis of Wien 133 cells (Figure 12A, B; set to 100%). The latter activity was not observed in the absence of serum, indicating cell lysis was Clq- dependent.

The single agent activity of IgGl-CAMPATH-lH-E430G was close to the activity of the mixture of IgGl-CAMPATH-lH-E430G + IgGl-llB8-E430G (Figure 12A). Introduction of self-oligomerization inhibiting mutation K439E into IgGl-CAMPATH- 1H-E430G resulted in strongly reduced cell lysis. An even further reduction of single agent activity was accomplished by introduction of mutations Y436N or Q438N in IgGl-CAMPATH-lH-E430G-K439E. Single agent activity was similarly suppressed after substitution of the Fc-Fc interaction enhancing mutation E430G by either E345K or E345R in IgGl-CAMPATH-lH-E430G-K439E-Y436N or IgGl-CAMPATH-lH-E430G- K439E-Q438N. In summary, single agent activity of IgGl-CAMPATH-lH with either of the Fc-Fc interaction enhancing mutations E430G, E345K or E345R can be abrogated by introduction of self-oligomerization inhibiting mutation K439E in combination with either Y436N or Q438N.

Antibody anti-CD20 IgGl-llB8-E430G shows intermediate single agent activity (Figure 12A). Introduction of self-oligomerization inhibiting mutation S440K abrogated the single agent activity of IgGl-llB8-E430G. Complete abrogation of single agent activity was also observed after introduction of self-oligomerization inhibiting mutation Y436K or Q438R in IgGl-llB8-E430G-S440K. Similar to the results described for IgGl-CAMPATH-lH, antibody variants with either of the Fc-Fc interaction enhancing mutations E345K or E345R instead of E430G resulted in similar abrogation of cell lysis as observed with antibody variants containing the E430G mutation. In summary, single agent activity of IgGl-llB8 with either of the Fc-Fc interaction enhancing mutations E430G, E345K or E345R can be abrogated by introduction of self-oligomerization inhibiting mutation S440K in combination with either Y436K or Q438R.

While only marginal single agent activity was observed by IgG 1-CAM PATH- 1H- E430G-K439E-Y436N and no single agent activity was observed by IgGl-llB8- E430G-S440K-Q438R, a mixture of these antibodies recovered CDC efficacy close to the level of a mixture of IgGl-CAMPATH-lH-E430G + IgGl-llB8-E430G (Figure 12A). Similar restoration of cell lysis was accomplished by mixing antibody variants in which the E430G mutation was replaced by the E345K or E345R mutation. As previously described in Example 11, only partial recovery of CDC efficacy was obtained by mixing IgGl-CAMPATH-lH-E430G-K439E-Y436N with IgGl-llB8- E430G-S440K-Y436K (Figure 12A). Similar results were obtained when the E430G mutation in the latter antibodies was replaced by the E345K mutation. A stronger recovery of CDC efficacy as compared with the variants harboring the E345K mutation was accomplished by mixing the same antibody variants in which the E430G mutation was replaced by E345R, which may be interesting when maximal potency is desired. While limited single agent activity was observed by IgGl- CAMPATH-1H-E345K-K439E-Q438N and by IgGl-llB8-E345K-S440K-Y436K, a mixture of these antibodies recovered ~65% of the CDC efficacy of positive control mixture of IgGl-CAMPATH-lH-E430G + IgGl-llB8-E430G (Figure 12A). Substituting E345K with E345R in both antibody variants in the mixture recovered ~ 80% CDC potency. Similar efficacy was observed for a mixture of IgGl-CAMPATH-lH-E345R- K439E-Q438N and IgGl-llB8-E345R-S440K-Q438R.

As described above, single agent activity could be reduced by introduction of mutation K439E in IgGl-CAMPATH-lH-E430G while single agent activity was completely abrogated by introduction of mutation S440K in IgGl-llB8 (Figure 12A, B). When the Fc-Fc interaction enhancing mutations K248E and T437R were introduced in IgGl-CAMPATH-lH instead of E430G, substantial single agent activity was observed, also when combined with the self-oligomerization inhibiting mutation K439E in combination with Y436N or Q438N (Figure 12B). No single agent activity was observed by IgGl-llB8 variants in which the K248E and T437R mutations were introduced together with S440K and either Y436K or Q438R. Although CDC efficacy could be enhanced by mixing antibodies containing the K248E and T437R mutations, this enhancement did not result in CDC efficacy as efficient as mixtures of antibodies containing any of the Fc-Fc interaction enhancing mutations E430G, E345K or E345R. Overall, these results show that recovery of CDC efficacy can be accomplished by mixing antibody variants harboring complementary self-oligomerization inhibiting mutations K439E, S440K, Y436K, Y436N, Q438N and/or Q438R, regardless of which of the largely functionally equivalent Fc-Fc interaction enhancing mutations E430G, E345K or E345R is included. Mixtures of two antibodies containing the Y436K and Y436N mutations only partially restored CDC efficacy. However, mixing variants of such antibodies containing the E345R mutation induced stronger recovery of CDC efficacy than antibody variants containing either the E345K or E430G mutation. Furthermore, a partial recovery of CDC efficacy could be accomplished by mixing antibody variants containing the K248E and T437R Fc-Fc interaction enhancing mutations, which was less efficient than mixtures of antibodies containing either of the E430G, E345K or E345R mutations.

Example 13: Analysis of selective CDC activity for mixtures of anti-CD52 and anti-CD20 antibody variants in different human IgG subclass backbones

In the previous Examples, it was described that the introduction of self- oligomerization inhibiting mutations in anti-CD20 and anti-CD52 IgGl antibodies resulted in selective co-dependent induction of target cell lysis. Here, we tested whether these principles also apply to other IgG subclasses and combinations of different IgG subclasses.

The VH sequences of anti-CD52 CAMPATH-1H were cloned in human IgGl, IgG2 and hinge-stabilized IgG4 (S228P) Fc backbones containing the E430G-K439E mutations, and the VH sequences of anti-CD20 11B8 were cloned in human IgGl, IgG2 and hinge-stabilized IgG4 (S228P) Fc backbones containing the E430G-S440K mutations. Different combinations of these anti-CD52 and anti-CD20 subclass variants with additional self-oligomerization inhibiting mutations Y436N, Y436K, Q438N, or Q438R were tested for selective CDC activity. An in vitro CDC assay using Wien 133 cells was performed with 20% NHS and antibody concentration series (final total IgG concentration range 0.01-40.0 pg/mL in 3.3-fold dilutions), essentially as described in Example 9. Cell lysis and relative AUC values were calculated from the number of Pi-positive cells as described in Example 9, from two experimental replicates. AUC was normalized to the values for negative control antibody IgGl-bl2 (0%) and for positive control IgGl-CAMPATH-lH-E430G + IgGl-llB8-E430G (100%).

As described in Examples 9, 10 and 11, the single agent activity of anti-CD20 IgGl- 11B8-E430G could be abrogated by introduction of self-oligomerization inhibiting mutation S440K in combination with either the Y436K or Q438R mutation. Introduction of the S440K, Y436K and Q438R mutations in IgG2 or IgG4 subclass backbones likewise resulted in abrogation of CDC efficacy. The CD52-targeting antibody variant IgGl-CAMPATH-lH-E430G-K439E-Y436N showed residual single agent activity, though much lower than the mixture of IgGl-CAMPATH-lH-E430G + IgGl-llB8-E430G. No CDC efficacy was observed for the IgG2 or IgG4 subclass backbone variants of this antibody (Figure 13) when used as a single agent, which is highly interesting if maximal selectivity for cells or tissues bound by both components is desired . A similar observation was made for IgG 1-CAM PATH- 1H- E430G-K439E-Q438N : while low single agent activity was observed for the IgGl subclass variant (Figure 11, Example 11) no single agent activity could be detected for the IgG2 or IgG4 subclass variants (Figure 13). Without being limited by theory, this may be explained by reduced Clq binding affinity of the IgG2 and IgG4 subclasses compared to IgGl.

Recovery of CDC efficacy could be attained by mixing IgG 1-CAM PATH- 1H-E430G- K439E-Y436N with IgGl-l lB8-E430G-S440K-Q438R, IgG2-l lB8-E430G-S440K-

Q438R or IgG4-l lB8-S228P-E430G-S440K-Q438R. CDC potency of the IgG2 and IgG4 combinations was lower compared to the mixture of the corresponding IgGl antibody variants (Figure 13). CDC efficacy could also be recovered by mixing IgG2- CAMPATH-1H-E430G-K439E-Y436N with IgG l-l lB8-E430G-S440K-Q438R, IgG2- 11B8-E430G-S440K-Q438R or IgG4-l lB8-S228P-E430G-S440K-Q438R, however with reduced CDC potency compared to the mixture of the corresponding IgGl antibody variants. Partial recovery of CDC efficacy could be accomplished by mixing IaG2-CAMPATH- lH-E430G-K439E-O438N with IgG2-l lB8-E430G-S440K-Y436K or IgG2- l lB8-E430G-S440K-Q438R with the latter combination showing more activity than the former combination. Partial recovery of CDC efficacy could also be attained by mixing IaG4-CAMPATH-lH-S228P-E430G-K439E-Y436N with IgG4-l lB8-S228P- E430G-S440K-Q438R or IgG l-l lB8-S440K-Q438R (Figure 13). No substantial recovery of CDC efficacy was observed by a mixture of IQG4-CAMPATH-1H-S228P- E430G-K439E-O438N and IgG4-l lB8-S228P-E430G-S440K-Q438R.

Consistent with the observations in Example 9, mixing IgGl-CAMPATH-lH-E430G- K439E-Y436N with IaGl-l lB8-E430G-S440K-Y436K did not result in recovery of CDC efficacy. Similarly, IgG2 subclass variants IgG2-CAMPATH-lH-E430G-K439E- Y436N + IgG2-l lB8-E430G-S440K-Y436K and IgG4 subclass variants IgG4- CAMPATH-1H-S228P-E430G-K439E-Y436N + IgG4- l lB8-S228P-E430G-S440K-

Y436K failed to recover substantial CDC activity.

When comparing IgG subclass combinations per individual subclass class, combinations of IgGl antibodies induced stronger co-dependent CDC efficacy than combinations of IgG2 antibodies, which in turn performed stronger than combinations of IgG4 antibodies. In addition, combinations of different IgG subclasses showed that combinations of an IgG l and an IgG2 antibody induced stronger co-dependent CDC efficacy than combinations of an IgGl and an IgG4 antibody, which in turn performed stronger than combinations of an IgG2 and IgG4 antibody. In conclusion, co-dependent CDC activity could be induced by antibodies derived from all tested IgG subclasses, both when using combinations of two antibodies derived from the same IgG subclass, as well as when derived from two different IgG subclasses.

Example 14: Analysis of selective CDC activity for mixtures of anti-CD52 and anti-CD20 antibody variants with FcyR-binding inhibiting mutation G237A

In Example 11 and subsequent Examples, it was described that the introduction of self-oligomerization inhibiting mutations in anti-CD20 and anti-CD52 IgGl antibodies resulted in selective co-dependent induction of target cell lysis. As an example of mutations that strongly suppress FcyR-binding and FcyR-mediated effector functions while having limited effect on Clq-binding or CDC by co-dependent antibodies, we tested the effect of further introducing mutation G237A. Only the effector functions sensitive to co-dependent hexamerization of the two components could be expected to recover after mixing, while both the single agents and the mixture would be expected to show severely inhibited FcyR-mediated effector functions.

To detect binding of IgGl-CAMPATH-lH and IgGl-llB8 antibody variants to dimeric FcyR variants, an FcyR binding assay was performed essentially as described in Example 8. Furthermore, In vitro CDC assays using Wien 133 cells were performed with 20% NHS and antibody concentration series, final concentration range 0.002- 40.0 pg/mL in 4-fold dilutions (Figure 14F) or 0.01-40.0 pg/mL in 3.3-fold dilutions (Figure 14G), essentially as described in Example 9. Cell lysis and relative AUC values were calculated from the number of Pi-positive cells as described in Example 9, from two experimental replicates. AUC was normalized to the values for negative control antibody IgGl-bl2 (0%) and for positive control IgGl-CAMPATH-lH-E430G (Figure 14F) or a mixture of IgGl-CAMPATH-lH-E430G + IgGl-llB8-E430G (100%; Figure 14G). No CDCactivity was observed after exposure of Wien 133 cells to the latter mixture in the absence of serum, indicating cell lysis was Clq-dependent (Figure 14G).

The wild-type IgG 1-CAM PATH- 1H antibody and variants thereof with introduced mutations E430G or E430G-K439E bound FcyRIIA allotype 131H, FcyRIIA allotype 131R, FcyRIIB, FcyRIIIA allotype 158F and FcyRIIIA allotype 158V (Figure 14A-E). The wild-type antibody IgGl-llB8 and the variant with introduced mutations E430G- S440K also showed binding to the tested FcyR variants. Antibody variant IgGl-llB8- E430G showed less efficient FcyR variant binding for reasons that were unclear, but retained substantial binding to high affinity variants FcyRIIA allotype 131H and FCYRIIIA allotype 158V. Binding to FcyR was completely abrogated by introduction of mutation G237A in all of the aforementioned antibody variants.

Efficient cell lysis was observed after exposing Wien 133 cells to IgGl-CAMPATH-No single agent activity was observed of IgGl-llB8-E430G-S440K-Q438R or IgGl- 11B8-E430G-S440K-Y436K, and the introduction of mutation G237A in these antibody variants did not affect this (Figure 14G). The residual single agent activity observed for IgGl-CAMPATH-lH-E430G-K439E-Y436N was efficiently abrogated by introduction of mutation G237A, which is highly interesting if maximal selectivity for cells or tissues bound by both components is desired. The mixture of IgGl- CAMPATH-1H-E430G-K439E-Y436N and IgGl-llB8-E430G-S440K-Q438R did induce efficient CDC, approaching the level of the mixture of IgGl-CAMPATH-lH-E430G and IgGl-llB8-E430G. Recovery of cell lysis was also observed after mixing IgGl- CAMPATH-1H-E430G-K439E-Y436N-G237A with IgGl-llB8-E430G-S440K-Q438R- G237A, but not after mixing with IgGl-llB8-E430G-S440K-Y436K-G237A, in line with results described in previous Examples.

The low single agent activity of IgGl-CAMPATH-lH-E430G-K439E-Q438N was eliminated upon introduction of mutation G237A (Figure 14). CDC efficacy could be restored to intermediate levels by mixing IgGl-CAMPATH-lH-E430G-K439E-Q438N- G237A with either IgGl-llB8-E430G-S440K-Y436K-G237A or IgGl-llB8-E430G- S440K-Q438R-G237A, but at antigen-saturating antibody concentrations approximately 80% lysis was observed. Overall, mixtures of antibody variants containing the G237A mutation show a relatively lower restoration of CDC efficacy than antibody variants without this mutation. Without being limited by theory, this may be explained by a modest inhibitory effect of mutation G237A on Clq binding.

In summary, these data demonstrate that the introduction of FcYR-binding inhibiting mutation G237A eliminates the residual single agent activity of antibody variants with self-oligomerization inhibiting mutations Y436N and Q438N. Recovery of CDC efficacy could be attained by mixing antibody variants with complementary self- oligomerization inhibiting mutations and the G237A mutation, albeit with lower efficiency than mixtures of complementary antibody variants without the G237A mutation. Example 15: Analysis of Clq binding by mixtures of anti-CD52 and anti- CD20 antibody variants with an FcyR-binding inhibiting mutation and enhanced Clq binding mutations

It was demonstrated in Example 14 that the introduction of FcYR-binding inhibiting mutation G237A resulted in elimination of single agent activity of anti-CD52 and anti-CD20 IgGl antibody variants with self-oligomerization inhibiting mutations. However, as compared to antibody variants without the G237A mutation, mixtures of complementary antibody variants containing the G237A mutation did not fully restore selective co-dependent CDC efficacy. Here, we tested whether the introduction of Clq binding enhancing mutations E333S or K326W-E333S in one antibody component of a mixture of two antibodies could compensate for the possibly reduced Clq binding of a G237A-containing antibody component.

An in vitro CDC assay using Wien 133 cells was performed with 20% NHS and antibody concentration series (final concentration range 0.01-40.0 pg/mL in 3.3-fold dilutions), essentially as described in Example 9. Cell lysis and relative AUC values were calculated from the number of Pi-positive cells as described in Example 9, from two experimental replicates. AUC was normalized to the values for negative control antibody IgGl-bl2 (0%) and for positive control IgGl-CAMPATH-lH-E430G + IgGl- 11B8-E430G (100%). The latter activity was not observed in the absence of serum, indicating cell lysis was Clq-dependent.

As described in Example 14, introduction of mutation G237A in IgG 1-CAM PATH- 1H- E430G-K439E variants with either the Y436N or Q438N mutation abrogated single agent activity (Figure 14, 15). The Clq-binding enhancing mutations E333S or K326W-E333S were introduced in anti-CD20 IgGl-llB8-E430G antibody variants to investigate the effects of this mutation on single agent CDC efficacy (Figure 15). While the introduction of E333S in IgGl-llB8-E430G-S440K-Q438R or IgGl-llB8- E430G-S440K-Y436K resulted in low CDC efficacy close to background levels, the introduction of the K326W-E333S mutations in the same antibodies resulted in intermediate single agent CDC efficacy.

Full recovery of CDC efficacy was observed after mixing IgGl-CAMPATH-lH-E430G- K439E-Y436N-G237A with IgGl-llB8-E430G-S440K-Q438R antibody variants containing either the E333S or K326W-E333S mutations (Figure 15). Partial recovery of CDC efficacy was attained by mixing IgGl-CAMPATH-lH-E430G-K439E-Y436N- G237A with IgGl-llB8-E430G-S440-Y436K-K326W-E333S and to a lesser extent with IgGl-llB8-E430G-S440-Y436K-E333S, in line with the results described in Example 9 and 11.

As described in both Example 14 and here, antibody variant IgGl-CAMPATH-lH- E430G-K439E-Q438N with mutation G237A did not show any single agent activity. Furthermore, mixtures of this antibody with IgGl-llB8-E430G-S440K variants containing either Y436K or Q438R and G237A did not fully restore CDC efficacy (Figure 14). However, CDC efficacy could be restored to levels closer to that of the positive control mixture by mixing IgGl-CAMPATH-lH-E430G-K439E-Q438N-G237A with variants of IgGl-llB8-E430G-S440K-Q438R or IgGl-llB8-E430G-S440K- Y436K containing either the E333S or K326W-E333S mutations (Figure 15), reaching absolute lysis levels of approximately 90% upon antigen saturation.

Taken together, the largest window of selectivity was attained by mixing one antibody harboring self-oligomerization inhibiting mutations and FcyR-binding inhibiting mutation G237A with an antibody harboring self-oligomerization inhibiting mutations and enhanced Clq-binding mutation E333S.

Example 16: Selectivity of CDC activity on Raji cells by mixed anti-CD37 IgGl-37-37-3 antibody variants with an E430G Fc-Fc interaction enhancing mutation and Fc-Fc self-oligomerization inhibiting mutations

The effect of self-oligomerization inhibiting mutations Y436K, Y436N, Q438N and Q438R on in vitro CDC efficacy on Raji cells was tested using mixtures of variants of anti-CD37 antibody IgGl-CD37-37-3 with an E430G Fc-Fc interaction enhancing mutation.

An in vitro CDC assay using Raji cells was performed with 20% NHS and antibody concentration series (final concentration range 0.01-40.0 pg/mL in 3.3-fold dilutions), essentially as described in Example 9. Burkitt's lymphoma cell line Raji was purchased from ATCC (Cat No. CCL-86). Cell lysis and relative AUC values were calculated from the number of Pi-positive cells as described in Example 9, from two experimental replicates. AUC was normalized to the values for negative control antibody IgGl-bl2 (0%) and for positive control IgGl-CAMPATH-lH-E430G + IgGl- CD37-37-3-E430G (100%).

Antibody IgGl-CD37-37-3-E430G on itself induced potent CDC efficacy on Raji cells, regardless of whether it was mixed with non-antigen binding antibody IgGl-bl2 or not (Figure 16). Although considerably reduced as compared with IgGl-CD37-37-3- E430G itself, residual single agent activity was observed upon introduction of self- oligomerization inhibiting mutations S440K, S440K-Y436K, S440K-Q438R, K439E- Y436N or K439E-Q438N in IgGl-CD37-37-3-E430G. CDC efficacy could be fully restored to the level of IgGl-CD37-37-3-E430G by mixing IgGl-CD37-37-3-E430G- K439E-Y436N with IgGl-CD37-37-3-E430G-S440K-Q438R, but not with IgGl-CD37- 37-3-E430G-S440K-Y436K, consistent with the results presented in Examples 9-15. Likewise, the reduced CDC efficacy of IgGl-CD37-37-3-E430G-K439E-Q438N could be partially restored by mixing with IgGl-CD37-37-3-E430G-S440K-Y436K or IgGl- CD37-37-3-E430G-S440K-Q438R.

Overall, these data show that CDC efficacy of IgGl-CD37-37-3-E430G on Raji cells could be partially abrogated by introduction of self-oligomerization inhibiting mutations K439E-Y436N, K439E-Q438N, S440K-Y436K or S440K-Q438R. Recovery of CDC efficacy was attained to varying extent by mixing, in order of strong to weak recovery, IgGl-CD37-37-3-E430G-K439E-Y436N with IgGl-CD37-37-3-E430G- S440K-Q438R, IgGl-CD37-37-3-E430G-K439E-Q438N with IgGl-CD37-37-3-E430G- S440K-Q438R or IgGl-CD37-37-3-E430G-K439E-Q438N with IgGl-CD37-37-3- E430G-S440K-Y436K.

Example 17: Selectivity of CDC activity on Raji cells by mixed anti-CD37 IgGl-37-37-3 and anti-CD20 IgGl-llB8 antibody variants with an E430G Fc-Fc interaction enhancing mutation and Fc-Fc self-oligomerization inhibiting mutations

The effect of self-oligomerization inhibiting mutations Y436K, Y436N, Q438N and Q438R on in vitro CDC efficacy on Raji cells was tested using mixtures of variants of anti-CD37 antibody IgGl-CD37-37-3 and anti-CD20 IgGl-llB8 with an E430G Fc-Fc interaction enhancing mutation.

An in vitro CDC assay using Raji cells was performed with 20% NHS and antibody concentration series (final concentration range 0.01-40.0 pg/mL in 3.3-fold dilutions), essentially as described in Example 9. Burkitt's lymphoma cell line Raji was purchased from ATCC (Cat No. CCL-86). Cell lysis and relative AUC values were calculated from the number of Pi-positive cells as described in Example 9, from two experimental replicates. AUC was normalized to the values for negative control antibody IgGl-bl2 (0%) and for positive control IgGl-CAMPATH-lH-E430G + IgGl- CD37-37-3-E430G (100%).

Both IgGl-CD37-37-3-E430G and IgGl-llB8-E430G demonstrated potent single agent CDC activity on Raji cells, albeit with lower efficiency than a mixture of said antibodies (Figure 17). Low single agent CDC activity was observed upon introduction of self-oligomerization inhibiting mutations S440K-Y436K or S440K- Q438R in IgGl-llB8-E430G and by introduction of mutations K439E-Y436N or K439E-Q438N in IgGl-CD37-37-3-E430G. CDC efficacy could be fully restored to the level of a mixture of IgGl-CD37-37-3-E430G and IgGl-llB8-E430G by mixing IgGl- CD37-37-3-E430G-K439E-Y436N with IgGl-llB8-E430G-S440K-Q438R, but not with IgGl-llB8-E430G-S440K-Y436K, consistent with the results presented in Examples 9-16. The reduced CDC efficacy of IgGl-CD37-37-3-E430G-K439E-Q438N could be partially restored by mixing with IgGl-llB8-E430G-S440K-Y436K or IgGl- 11B8-E430G-S440K-Q438R.

Overall, these data show that CDC efficacy of IgGl-CD37-37-3-E430G on Raji cells could be partially abrogated by introduction of self-oligomerization inhibiting mutations K439E-Y436N or K439E-Q438N. Similarly, CDC efficacy of IgGl-llB8- E430G on Raji cells could be partially abrogated by introduction of self- oligomerization inhibiting mutations S440K-Y436K or S440K-Q438R. Recovery of CDC efficacy was attained to varying extent by mixing, in order of strong to weak recovery, IgGl-CD37-37-3-E430G-K439E-Y436N with IgGl-llB8-E430G-S440K- Q438R, IgGl-CD37-37-3-E430G-K439E-Q438N with IgGl-llB8-E430G-S440K-

Q438R, or IgGl-CD37-37-3-E430G-K439E-Q438N with IgGl-llB8-E430G-S440K- Y436K.

Example 18: Selectivity of CDC activity on Raji cells by mixed anti-CD52 IgGl-CAMPATH-lH and anti-CD37 IgGl-37-37-3 antibody variants with an E430G Fc-Fc interaction enhancing mutation and Fc-Fc self-oligomerization inhibiting mutations

The effect of self-oligomerization inhibiting mutations Y436K, Y436N, Q438N, Q438R, K439E and S440K on in vitro CDC efficacy on Raji cells was tested using mixtures of variants of anti-CD52 IgG 1-CAM PATH- 1H and anti-CD37 IgGl-37-37-3 with an E430G Fc-Fc interaction enhancing mutation.

An in vitro CDC assay using Raji cells was performed with 20% NHS and antibody concentration series (final concentration range 0.01-40.0 pg/mL in 3.3-fold dilutions), essentially as described in Example 9. Burkitt's lymphoma cell line Raji was purchased from ATCC (Cat No. CCL-86). Cell lysis and relative AUC values were calculated from the number of Pi-positive cells as described in Example 9, from two experimental replicates. AUC was normalized to the values for negative control antibody IgGl-bl2 (0%) and for positive control IgGl-CAMPATH-lH-E430G + IgGl- CD37-37-3-E430G ( 100%). Antibody variant IgG 1-CAM PATH- 1H-E430G showed low to intermediate single agent CDC activity on Raji cells, while the single agent CDC activity of IgGl-CD37-37-3- E430G reached almost 80% of the activity induced by the mixture of IgGl- CAMPATH-1H-E430G and IgGl-CD37-37-3-E430G (Figure 18). Introduction of self- oligomerization inhibiting mutation K439E in IgGl-CAMPATH-lH-E430G completely abrogated single agent CDC activity, while introduction of mutation S440K in IgGl- CD37-37-3-E430G reduced single agent CDC activity. By mixing IgGl-CAMPATH-lH- E430G-K439E and IgGl-CD37-37-3-E430G-S440K, a modest increase in CDC activity could be attained to approximately 40% of the level of the positive control mixture. Introduction of mutation Y436K, Y436N, Q438N or Q438R in either IgGl-CD37-37-3- E430G-S440K or IgGl-CAMPATH-lH-E430G-K439E did not significantly affect single agent CDC activity.

After mixing antibody variants of IgGl-CAMPATH-lH-E430G and IgGlCD37-37-3- E430G, CDC could only be partially recovered by a mixture of IgG 1-CAM PATH- 1H- E430G-K439E-Y436N and IgGl-CD37-37-3-E430G-S440K-Q438R, and not by mixing IgG 1-CAM PATH- 1H-E430G-K439E-Y436N with IgGl-CD37-37-3-E430G-S440K- Y436K, mixing IgGl-CAMPATH-lH-E430G-K439E-Q438N with IgGl-CD37-37-3- E430G-S440K-Y436K or mixing IgGl-CAMPATH-lH-E430G-K439E-Q438N with IgGl- CD37-37-3-E430G-S440K-Q438R.

Consistent with these observations, when an S440K and an additional self- oligomerization mutation were introduced in the IgGl-CAMPATH-lH-E430G antibody instead of the IgGl-CD37-37-3-E430G antibody, a partial recovery of CDC activity could only be attained by mixing IgGl-CAMPATH-lH-E430G-S440K-Q438R with IgGl-CD37-37-3-E430G-K439E-Y436N, and not by mixing IgG 1-CAM PATH- 1H- E430G-S440K-Y436K with IgGl-CD37-37-3-E430G-K439E-Y436N, mixing IgGl- CAMPATH-1H-E430G-S440K-Y436K with IgGl-CD37-37-3-E430G-K439E-Q438N or mixing IgGl-CAMPATH-lH-E430G-S440K-Q438R with IgGl-CD37-37-3-E430G- K439E-Q438N.

Overall, the results presented here indicate that mixtures of IgGl-CAMPATH-lH- E430G and IgGl-CD37-37-3-E430G antibody variants harboring self-oligomerization inhibiting mutations induced partial recovery of CDC efficacy in Raji cells, only when an antibody variant harboring the K439E-Y436N mutations was mixed with an antibody variant harboring the S440K-Q438R mutations. These effects were observed regardless of whether the aforementioned combinations of mutations were introduced in the IgGl-CAMPATH-lH-E430G or IgGl-CD37-37-3-E430G antibody variants.

Example 19: Selective DR5 agonist activity of a mixture of two noncrossblocking anti-DR5 antibodies with an E430G Fc-Fc interaction enhancing mutation and self-oligomerization inhibiting mutations on BxPC-3 cells

The mixture of the two non-crossblocking anti-death receptor 5 (DR5) antibodies IgGl-DR5-01-G56T-E430G + IgGl-DR5-05-E430G acts as a DR5 agonist inducing killing of DR5-positive cancer cells (WO17093447). Here, a viability assay was performed to study whether the introduction of self-oligomerization inhibiting mutations Y436K, Y436N, Q438N and Q438R in mixed DR5-targeting antibody variants results in co-dependent cytotoxicity on BxPC-3 pancreatic cancer cells (ATCC, Cat No. CRL-1687), which express low levels of DR5 (data not shown).

BxPC-3 cells were harvested by trypsinization and passed through a cell strainer. Cells were pelleted by centrifugation for 5 minutes at 1,200 rpm and resuspended in culture medium at a concentration of 1.1x10 ^s cells/mL (RPMI 1640 medium (ATCC modification), Life Technologies Cat No. A10491-01+ 10% DBSI (Life Technologies Cat No. 20371). 45 pL of the single cell suspensions (5,000 cells/well) were seeded in polystyrene 96-well flat-bottom plates (Greiner Bio-One, Cat No. 655180) and allowed to adhere overnight at 37°C. The next day, 50 pL samples of an antibody dilution series (final concentration range 0.003-20 pg/mL in 3-fold dilutions) and 24 pL purified human Clq stock solution (Quidel, Cat No. A400, 2.9 pg/mL final concentration) were added and incubated for 3 days at 37°C. As a positive control, cells were incubated with 5 pM staurosporine (Sigma Aldrich, Cat No. S6942). The viability of the cell cultures was determined in a CellTiter-Glo luminescence cell viability assay (Promega, Cat No. G755A) that quantifies the ATP present, which is an indicator of metabolically active cells. From the kit, 12 pL Luciferin Solution Reagent was added per well. Next, plates were incubated for 1.5 hours at 37°C. 100 pL supernatant was transferred to a white OptiPlate-96 (Perkin Elmer, Cat No. 6005290) and luminescence was measured on an EnVision Multilabel Reader (PerkinElmer). Data were analyzed using GraphPad Prism 7 and plotted as cell viability at 20 pg/ml antibody concentration. The percentage viable cells was calculated using the following formula : % viable cells = [(luminescence antibody sample - luminescence staurosporine sample)/(luminescence no antibody sample - luminescence staurosporine sample)]*100. No cytotoxicity was observed after exposure of BxPC-3 cells to negative controls medium or IgGl-bl2 (Figure 19). Also, no single agent activity was observed after exposing BxPC-3 cells to 20 pg/ml of either IgGl-DR5-01-G56T-E430G or IgGl-DR5- 05-E430G. In contrast, strong cytotoxicity was induced by a mixture of 20 pg/ml IgGl-hDR5-01-G56T-E430G and IgGl-hDR5-05-E430G, resulting in a cell viability of approximately 22%. Similarly, strong cytotoxicity was induced by a mixture of the same antibodies in which either of the self-oligomerization inhibiting mutations K439E and S440K were introduced, while the single components did not induce any cytotoxicity.

No single agent cytotoxicity was observed by antibody variants of IgGl-DR5-01- G56T-E430G and IgGl-DR5-05-E430G in which either of the K439E or S440K mutations, in combination with either of the Y436K, Y436N, Q438N or Q438R mutations were introduced. However, the potency to induce cytotoxicity could be recovered by mixing, in order of strong to weak recovery, IgGl-DR5-01-G56T- E430G-K439E-Y436N with IgGl-DR5-05-E430G-S440K-Q438R, IgGl-DR5-01-G56T- E430G-K439E-Q438N with IgGl-DR5-05-E430G-S440K-Q438R, or IgGl-DR5-01- G56T-E430G-K439E-Q438N with IgGl-DR5-05-E430G-S440K-Y436K. In line with the results described in Examples 9 - 17, no recovery of cytotoxicity was observed by mixing IgGl-DR5-01-G56T-E430G-K439E-Y436N with IgGl-DR5-05-E430G-S440K- Y436K.

In summary, these data show that DR5-targeting antibody variants with introduced self-oligomerization inhibiting mutations K439E, S440K, Y436K, Y436N, Q438N and Q438R do not induce single agent cytotoxicity of BxPC-3 cells, while cytotoxicity is restored by mixing complementary DR5-targeting antibody variants. Notably, the data presented in this Example represent a different mechanism of action compared to the data the described in Examples 9 - 17.

Example 20: Selectivity of CDC activity on Raji cells by mixed anti-CD20 IgGl-7D8 antibody variants with an E430G Fc-Fc interaction enhancing mutation and Fc-Fc self-oligomerization inhibiting mutations

The effect of self-oligomerization inhibiting mutations Y436K, Y436N, Q438N, Q438R, K439E, and S440K on in vitro CDC efficacy on Raji cells was tested using mixtures of variants of anti-CD20 IgGl-7D8 with an E430G Fc-Fc interaction enhancing mutation, as opposed to Example 10 in which antibody variants of anti-CD20 IgGl- 11B8 were tested. An in vitro CDC assay using Raji cells was performed with 20% NHS and antibody concentration series (final concentration range 0.01-40.0 pg/mL in 3.3-fold dilutions), essentially as described in Example 9. Burkitt's lymphoma cell line Raji was purchased from ATCC (Cat No. CCL-86). Cell lysis and relative AUC values were calculated from the number of Pi-positive cells as described in Example 9, from two experimental replicates. AUC was normalized to the values for negative control antibody IgGl-bl2 (0%) and for positive control IgGl-CAMPATH-lH-E430G + IgGl- 7D8-E430G (100%).

Antibody IgGl-7D8-E430G on itself induced potent CDC efficacy on Raji cells, regardless of whether it was mixed with non-antigen binding antibody IgGl-bl2 or not (Figure 20). Antibody variants of IgGl-7D8-E430G in which the self- oligomerization inhibiting mutations K439E-Y436N, K439E-Q438N, S440K-Y436K or S440K-Q438R were introduced demonstrated substantial single agent activity. CDC efficacy could be fully restored to the level of IgGl-7D8-E430G by mixing IgGl-7D8- E430G-K439E-Y436N with IgGl-7D8-E430G-S440K-Q438R, but not with IgGl-7D8- E430G-S440K-Y436K, consistent with the results presented in Examples 9-17. The reduced CDC efficacy of IgGl-7D8-E430G-K439E-Q438N could be partially restored by mixing with IgGl-7D8-E430G-S440K-Y436K or IgGl-7D8-E430G-S440K-Q438R. Overall, these data show that CDC efficacy of IgGl-7D8-E430G on Raji cells could be partially abrogated by introduction of self-oligomerization inhibiting mutations K439E-Y436N or K439E-Q438N. Similarly, CDC efficacy of IgGl-7D8-E430G on Raji cells could be partially abrogated by introduction of self-oligomerization inhibiting mutations S440K-Y436K or S440K-Q438R. Recovery of CDC efficacy was attained to varying extent by mixing, in order of strong to weak recovery, IgGl-7D8-E430G- K439E-Y436N with IgGl-7D8-E430G-S440K-Q438R, IgGl-7D8-E430G-K439E-Q438N with IgGl-7D8-E430G-S440K-Q438R, or IgGl-7D8-E430G-K439E-Q438N with IgGl- 7D8-E430G-S440K-Y436K.

Example 21: Selectivity of CDC activity on Wien 133 cells after titrating components of a mixture of anti-CD52 IgG 1-CAM PATH- 1H and anti-CD20 IgGl-llB8 antibody variants with an E430G Fc-Fc interaction enhancing mutation and self-oligomerization inhibiting mutations

In the previous Examples, antibody variants harboring an Fc-Fc interaction enhancing mutation in combination with one or more self-oligomerization inhibiting mutations were mixed in a 1 : 1 ratio. Here, we tested whether selective co- dependent CDC activity was also attained by mixing two antibody variants at different ratios.

An in vitro CDC assay using Wien 133 cells was performed with 20% NHS, essentially as described in Example 9. Single antibodies were titrated in 5-fold dilutions (final concentration range 0.0003-20.0 pg/mL) . When antibody mixtures were applied, one component was titrated (final concentration range 0.0003-20.0 pg/mL in 5-fold dilutions) and the other component was used at a fixed concentration of 20 or 2 pg/mL. Cell lysis was calculated from the number of Pi-positive cells as described in Example 9, from two experimental replicates.

Efficient CDC activity on Wien 133 cells was induced by a titrated mixture (1 : 1 ratio) of IgG 1-CAM PATH- 1H-E430G-K439E-Q438N and IgGl-l lB8-E430G-S440K-Y436K (Figure 21A), consistent with the results in Examples 11, 14, and 15. When CD20 was saturated with 20 pg/mL IgGl-l lB8-E430G-S440K-Y436K, >60% lysis was already detected in the presence of 0.16 pg/mL IgGl-CAMPATH-lH-E430G-K439E- Q438N. Likewise, saturating CD52 with 2 pg/mL IgGl-CAMPATH-lH-E430G-K439E- Q438N yielded >60% lysis in the presence of 0.16 pg/mL IgGl-l lB8-E430G-S440K- Y436K. In contrast, low CDC efficacy close to background levels was observed for a mixture of titrated IgGl-CAMPATH- lH-E430G-K439E-Q438N and either 20 pg/mL of non-antigen binding IgGl-bl2-E430G-S440K-Y436K or IgGl-bl2. Upon mixing 20 pg/mL IgGl-l lB8-E430G-S440K-Y436K with 20 pg/mL of either IgGl-bl2-E430G- K439E-Q438N or IgGl-bl2, no CDC activity was observed.

Similar patterns of CDC activity were observed for a titrated mixture (1:1 ratio) of IgG 1-CAM PATH- 1H-E430G-K439E-Q438N and IgGl-l lB8-E430G-S440K-Q438R (Figure 21B), consistent with Examples 11, 14, and 15. When saturating CD20 using 20 pg/mL IgGl-l lB8-E430G-S440K-Q438R, 0.16 pg/mL IgGl-CAMPATH-lH-E430G- K439E-Q438N sufficed to induce >60% lysis. When CD52 was saturated with a fixed concentration of 2 pg/mL of IgGl-CAMPATH-lH-E430G-K439E-Q438N , 0.04 pg/mL IgGl- l lB8-E430G-S440K-Q438R already sufficed to induce >50% lysis. Low CDC efficacy, close to background levels, was observed for a mixture of titrated IgGl- CAMPATH-1H-E430G-K439E-Q438N and either 20 pg/mL of non-antigen binding IgGl-bl2-E430G-S440K-Q438R or IgGl-bl2. Upon mixing 20 pg/mL IgG l-l lB8- E430G-S440K-Q438R with 20 pg/mL of either IgGl-bl2-E430G-K439E-Q438N or IgGl-bl2, no CDC activity was observed.

From these data, it can be concluded that efficient CDC activity could still be induced by complementary antibody variants harboring an Fc-Fc interaction enhancing mutation and self-oligomerization inhibiting mutations when mixed at different antibody ratios in which either of the components was present at >50-fold excess relative to the other component.

Example 22: Selectivity of CDC activity on Wien 133 cells through antigenbinding independent hexamerization of mixed antibody variants with an E430G Fc-Fc interaction enhancing mutation and Fc-Fc self-oligomerization inhibiting mutations

In the previous Examples, it was demonstrated that single agent CDC activity of antigen-binding antibody variants harboring an Fc-Fc interaction enhancing mutation could be reduced or abrogated by introducing self-oligomerization inhibiting mutations. Recovery of CDC efficacy was observed after mixing complementary antigen-binding antibody variants harboring self-oligomerization inhibiting mutations. Here, we tested whether co-dependent hexamerization could also be induced by mixtures of antigen-binding and non-antigen-binding antibody variants harboring said mutations.

An in vitro CDC assay using Wien 133 cells was performed with 20% NHS and antibody concentration series (final concentration range 0.01-40.0 pg/mL in 3.3-fold dilutions), essentially as described in Example 9. Cell lysis and relative area under the curve (AUC) values were calculated from the number of Pi-positive cells as described in Example 9, from two experimental replicates. AUC was normalized to the values for negative control antibody IgGl-bl2 (0%) and for positive control IgGl- CAMPATH-1H-E430G (100%).

Efficient CDC activity was observed after exposing Wien 133 cells to IgGl-CAMPATH- 1H-E430G as a single agent (Figure 22A). The introduction of additional self- oligomerization inhibiting mutations K439E or S440K reduced the single agent CDC efficacy, while CDC efficacy was fully restored by mixing IgGl-CAMPATH-lH-E430G- K439E and IgGl-CAMPATH-lH-E430G-S440K. A slight increase in CDC activity was observed when IgGl-CAMPATH-lH-E430G-K439E was mixed with non-antigen binding antibody variant IgGl-bl2-E430G-S440K, while similar activity was observed for a mixture of IgGl-bl2-E430G-K439E and IgGl-CAMPATH-lH-E430G-S440K. Apparently, upon binding to an antigen by at least one of the components in a mixture, non-antigen binding antibody variants harboring complementary mutations could be recruited from solution. At the highest antibody concentration tested (40 pg/ml), CDC by mixtures of one antigen-binding and one non-antigen binding antibody variant harboring the E430G mutation and self-oligomerization inhibiting mutations K439E or S440K was as efficient as a mixture of IgG 1-CAM PATH- 1H- E430G-K439E and IgGl-CAMPATH-lH-E430G-S440K (Figure 22B) although the latter mixture was superior at lower concentrations as shown by its higher AUC value in Figure 22A.

Introduction of additional self-oligomerization inhibiting mutations Y436K, Y436N, Q438N or Q438R in IgGl-CAMPATH-lH-E430G resulted in a reduction of single agent CDC activity (Figure 22C). CDC efficacy was increased to variable extent by mixing IgG 1-CAM PATH- 1H-E430G-K439E-Y436N with IgGl-bl2-E430G-S440K antibody variants harboring complementary mutations or by mixing IgGl-bl2-E430G-K439E- Y436N with IgGl-CAMPATH- lH-E430G-S440K antibody variants harboring complementary mutations (Figure 22C). Especially the mixture of IgGl-CAMPATH- 1H-E430G-K439E-Y436N + IgGl-CAMPATH-lH-E430G-S440K-Q438R showed potent maximal lysis when the CD52-binding specificity of either component was replaced with bl2 (Figure 22D), indicating that the selectivity of this mixture for only cells bound by both antibodies may be compromised at 40 pg/ml antibody concentration. In contrast, mixture IgGl-CAMPATH-lH-E430G-K439E-Y436N + IgG 1-CAM PATH- 1H- E430G-S440K-Q438N showed similar maximal activity and relative potency, but remained largely dependent on antigen binding by IgGl-CAMPATH-lH-E430G- K439E-Y436N even at 40 pg/ml antibody concentration.

CDC efficacy was increased to a limited extent by mixing IgGl-CAMPATH-lH-E430G- K439E-Q438N with IgGl-bl2-E430G-S440K antibody variants harboring complementary mutations or by mixing IgGl-bl2-E430G-K439E-Q438N with IgGl- CAMPATH-1H-E430G-S440K antibody variants harboring complementary mutations (Figure 22E) . Both when considering CDC efficacy (22E) and maximally induced cell lysis (at 40 pg/ml antibody concentration; Figure 22F), mixtures with two antigen- bound components remained substantially more active than mixtures with only one antigen-bound component.

These data indicate that introduction of self-oligomerization inhibiting mutations K439E or S440K in combination with Y436K, Y436N, Q438N or Q438R in IgGl antibody variants harboring the Fc-Fc interaction enhancing mutation E430G could result in residual CDC efficacy on Wien 133 cells when one of the antibody components did not bind the Wien 133 cells. In these experiments, the antibody mixtures displaying the largest difference in CDC activity between that induced by two antigen-bound components compared to that induced by one antigen-bound component were: antibodies harboring E430G-K439E-Q438N mutations mixed with antibodies harboring mutations E430G-S440K-Y436K, E430G-S440K-Q438R, or E430G-S440K-Y436N ; and antibodies harboring E430G-K439E-Y436N mutations mixed with antibodies harboring mutations E430G-S440K-Q438N.