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Title:
METHODS OF TREATMENT
Document Type and Number:
WIPO Patent Application WO/2021/092060
Kind Code:
A1
Abstract:
The disclosure relates to multispecific antibodies against BCMA for use in the treatment of lymphoma, such as non-Hodgkin's Lymphoma (NHL).

Inventors:
HAGNER PATRICK (US)
GANDHI ANITA (US)
CHIU HSILING (US)
WALDMAN MICHELLE (US)
Application Number:
PCT/US2020/058943
Publication Date:
May 14, 2021
Filing Date:
November 04, 2020
Export Citation:
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Assignee:
ENGMAB SARL (CH)
HAGNER PATRICK (US)
GANDHI ANITA (US)
CHIU HSILING (US)
WALDMAN MICHELLE (US)
International Classes:
A61K39/00; A61K39/395; A61P17/00; A61P35/00
Domestic Patent References:
WO2017031104A12017-02-23
Foreign References:
US20160176973A12016-06-23
US20180118842A12018-05-03
Attorney, Agent or Firm:
MARTINEK, Sebastian et al. (US)
Download PDF:
Claims:
Claims

1. A multispecific antibody that binds to BCMA and an antigen that promotes activation of one or more T cells for use in treating lymphoma.

2. The multispecific antibody for use according to claim 1, wherein the antigen that promotes activation of one or more T cells is selected from the group consisting of CD3, TCRa, TCRp. TCRy, TCR ICOS,

CD28, CD27, HVEM, LIGHT, CD40, 4-1BB, 0X40, DR3, GITR, CD30, TIM1, SLAM, CD2, or CD226.

3. The multispecific antibody for use according to claim 1 or 2, wherein the antigen that promotes activation of one or more T cells is CD3. 4. The multispecific antibody for use according to any one of claims 1-3, wherein the multispecific antibody is a bispecific antibody, optionally wherein the bispecific antibody comprises two Lab fragments of an anti-BCMA antibody, one Lab fragment of an anti-CD3 antibody, and one Pc portion and the bispecific antibody is in the format BCMA Lab - Pc - CD3 Lab - BCMA Lab.

5. The multispecific antibody for use according to any one of claims 1-4, wherein the multispecific antibody comprises an anti-BCMA antibody, or antigen binding fragment thereof, which comprises a VH and a VL selected from the group consisting of: a) a VH region of SEQ ID NO: 10 and a VL region of SEQ ID NO: 12, b) a VH region of SEQ ID NO: 10 and a VL region of SEQ ID NO: 13, c) a VH region of SEQ ID NO: 10 and a VL region of SEQ ID NO: 14, d) a VH region of SEQ ID NO:38 and a VL region of SEQ ID NO: 12, e) a VH region of SEQ ID NO:39 and a VL region of SEQ ID NO: 12, f) a VH region of SEQ ID NO:40 and a VL region of SEQ ID NO: 12, or g) a VH region of SEQ ID NO:9 and a VL region of SEQ ID NO: 11.

6. The multispecific antibody for use according to any one of claims 1-5, wherein the multispecific antibody comprises an anti-CD3 antibody, or antigen binding fragment thereof, which comprises a VH region of SEQ ID NO:7 and a VL region of SEQ ID NO:8.

7. The multispecific antibody for use according to claim 4, wherein the bispecific antibody comprises a heavy and light chain set of the polypeptides set forth in SEQ ID NO:48, SEQ ID NO:55, SEQ ID NO:56, and SEQ ID NO: 57 (x2).

8. The multispecific antibody for use according to any one of claims 1-7, wherein the lymphoma is Non- Hodgkin’s lymphoma (NHL).

9. The multispecific antibody for use according to any one of claims 1-8, wherein the lymphoma is diffuse large B-cell lymphoma (DLBCL). 10. The multispecific antibody for use according to claim 9, wherein the DLBCL is front line DLBCL.

11. The multispecific antibody for use according to claim 9, wherein the DLBCL is relapsed or refractory DLBCL.

12. The multispecific antibody for use according to any one of claims 1-8, wherein the lymphoma is follicular lymphoma. 13. The multispecific antibody for use according to claim 12, wherein the follicular lymphoma is front line follicular lymphoma.

14. The multispecific antibody for use according to claim 12, wherein the follicular lymphoma is relapsed or refractory follicular lymphoma.

15. The multispecific antibody for use according to any one of claims 1-8, wherein the lymphoma is mantle cell lymphoma, optionally wherein the mantle cell lymphoma is front line mantle cell lymphoma or relapsed or refractory mantle cell lymphoma.

Description:
METHODS OF TREATMENT

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of European Application No. EP19207296.5 filed November 5, 2019, the content of which is incorporated herein by reference in its entirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

This application incorporates by reference a Sequence Listing submitted with this application as text file entitled “14247-609-228_Sequence_Listing8.TXT” created on November 4, 2020 and having a size of 68,049 bytes.

FIELD OF THE INVENTION

The present invention relates to multispecific antibodies against BCMA for use in the treatment of diseases, such as lymphoma, e.g. non-Hodgkin’s Lymphoma (NHL).

BACKGROUND

Lymphoma is a group of blood cancers that develop from lymphocytes. Non-Hodgkin’s lymphoma (NHL) is a heterogeneous collection of lymphomas that can be classified into two major subgroups: aggressive lymphomas and indolent lymphomas. Diffuse large B-cell lymphoma (DLBCL) is the most prevalent subtype of aggressive lymphomas and follicular lymphoma (FL) is the largest constituent of indolent lymphomas. Existing therapies for NHL are not always effective and/or can have undesirable side effects. There is therefore a need for further therapies for the treatment of NHL.

SUMMARY

The present invention relates to methods of treating a patient having a lymphoma (e.g. Non- Hodgkin’s lymphoma (NHL)) using multispecific (e.g. bispecific) antibodies that bind to BCMA and an antigen that promotes activation of one or more T cells (e.g. CD3).

Thus, in one aspect, the present invention provides a method for treating lymphoma (e.g. NHL) in a patient (e.g. a human), the method comprising administering a multispecific (e.g. bispecific) antibody that binds to BCMA and an antigen that promotes activation of one or more T cells (e.g. CD3) to the patient.

In another aspect, the present invention provides a multispecific (e.g. bispecific) antibody that binds to BCMA and an antigen that promotes activation of one or more T cells (e.g. CD3) for use in treating a lymphoma (e.g. NHL) in a patient (e.g. a human).

In particularly preferred embodiments, the lymphoma is a B cell lymphoma, e.g. B cell NHL. In some embodiments, the lymphoma is DLBCL, including frontline DLBCL and/or relapsed or refractory DLBCL. In some embodiments, the lymphoma is follicular lymphoma, including frontline follicular lymphoma and/or relapsed or refractory follicular lymphoma. In some embodiments, the lymphoma is mantle cell lymphoma, including frontline mantle cell lymphoma and/or relapsed or refractory mantle cell lymphoma.

In one aspect, the present invention provides a method for treating B cell lymphoma (e.g. B cell NHL) in a patient (e.g. a human), the method comprising administering a multispecific (e.g. bispecific) antibody that binds to BCMA and an antigen that promotes activation of one or more T cells (e.g. CD3) to the patient.

In another aspect, the present invention provides a multispecific (e.g. bispecific) antibody that binds to BCMA and an antigen that promotes activation of one or more T cells (e.g. CD3) for use in treating a B cell lymphoma (e.g. B cell NHL) in a patient (e.g. a human).

In some embodiments, the multispecific (e.g. bispecific) antibody comprises an anti-BCMA antibody, or antigen binding fragment thereof, comprising a CDR3H region of SEQ ID NO: 17 and a CDR3L region of SEQ ID NO:20 and a CDR1H, CDR2H, CDR1L, and CDR2L region combination selected from the group of: a) CDR1H region of SEQ ID NO:21 and CDR2H region of SEQ ID NO:22, CDR1L region of SEQ ID NO: 23, and CDR2L region of SEQ ID NO: 24, b) CDR1H region ofSEQ ID NO:21 and CDR2H region of SEQ ID NO:22, CDR1L region of SEQ ID NO:25, and CDR2L region of SEQ ID NO:26, c) CDR1H region of SEQ ID NO:21 and CDR2H region of SEQ ID NO:22, CDR1L region of SEQ ID NO:27, and CDR2L region of SEQ ID NO:28, d) CDR1H region of SEQ ID NO: 29 and CDR2H region of SEQ ID NO: 30, CDR1L region of SEQ ID NO: 31, and CDR2L region of SEQ ID NO: 32, e) CDR1H region of SEQ ID NO:34 and CDR2H region of SEQ ID NO:35, CDR1L region of SEQ ID NO: 31, and CDR2L region of SEQ ID NO: 32, f) CDR1H region of SEQ ID NO:36 and CDR2H region of SEQ ID NO:37, CDR1L region of SEQ ID NO: 31, and CDR2L region of SEQ ID NO: 32, and g) CDR1H region of SEQ ID NO: 15 and CDR2H region of SEQ ID NO: 16, CDR1L region of SEQ ID NO: 18, and CDR2L region of SEQ ID NO: 19.

In some embodiments, the anti-BCMA antibody, or antigen binding fragment thereof, comprises a VH and a VL selected from the group consisting of: a) a VH region of SEQ ID NO: 10 and a VL region of SEQ ID NO: 12, b) a VH region of SEQ ID NO: 10 and a VL region of SEQ ID NO: 13, c) a VH region of SEQ ID NO: 10 and a VL region of SEQ ID NO: 14, d) a VH region of SEQ ID NO:38 and a VL region of SEQ ID NO: 12, e) a VH region of SEQ ID NO:39 and a VL region of SEQ ID NO: 12, f) a VH region of SEQ ID NO:40 and a VL region of SEQ ID NO: 12, or g) a VH region of SEQ ID NO: 9 and a VL region of SEQ ID NO: 11.

In particularly preferred embodiments, the anti-BCMA antibody, or antigen binding fragment thereof, comprises a VH region of SEQ ID NO: 10 and a VL region of SEQ ID NO: 14.

In some embodiments, the antigen that promotes activation of one or more T cells is selected from the group consisting of CD3, TCRa, TCR , TCRy, TCR , ICOS, CD28, CD27, HVEM, LIGHT, CD40, 4-1BB, 0X40, DR3, GITR, CD30, TIM1, SLAM, CD2, or CD226. In particularly preferred embodiments, the antigen that promotes activation of one or more T cells is CD3.

In some embodiments, the multispecific (e.g. bispecific) antibody comprises an anti-CD3 antibody, or antigen binding fragment thereof, comprising a variable domain VH comprising the heavy chain CDRs of SEQ ID NO: 1, 2 and 3 as respectively heavy chain CDR1H, CDR2H and CDR3H and a variable domain VL comprising the light chain CDRs of SEQ ID NO: 4, 5 and 6 as respectively light chain CDR1L, CDR2L and CDR3L. In some embodiments, the anti-CD3 antibody, or antigen binding fragment thereof, comprises a VH region of SEQ ID NO: 7 and a VL region of SEQ ID NO:8.

In particularly preferred embodiments, the multispecific (e.g. bispecific) antibody comprises an anti-BCMA antibody, or antigen binding fragment thereof, comprising a VH region of SEQ ID NO: 10 and a VL region of SEQ ID NO: 14, and an anti-CD3 antibody, or antigen binding fragment thereof, comprising a VH region of SEQ ID NO:7 and a VL region of SEQ ID NO: 8.

In some embodiments, the multispecific antibody is a bispecific antibody. In some embodiments, the bispecific antibody is bivalent (the 1+1 format). In some embodiments, the bivalent bispecific antibody has the format: CD3 Fab - BCMA Fab (i.e. when no Fc is present). Alternatively, the bivalent bispecific antibody may have the format: Fc - CD3 Fab - BCMA Fab; Fc- BCMA Fab - CD3 Fab; or BCMA Fab - Fc - CD3 Fab (i.e. when an Fc is present). In preferred embodiments, the bivalent bispecific antibody has the format BCMA Fab - Fc - CD3 Fab. In some embodiments, the bispecific antibody is trivalent (the 2+1 format). In some embodiments, the trivalent bispecific antibody has the format: CD3 Fab - BCMA Fab - BCMA Fab; or BCMA Fab - CD3 Fab - BCMA Fab (i.e. when no Fc is present). Alternatively, the trivalent bispecific antibodies may have the format: BCMA Fab - Fc - CD3 Fab - BCMA Fab; BCMA Fab - Fc - BCMA Fab - CD3 Fab; or CD3 Fab - Fc - BCMA Fab - BCMA Fab (i.e. when an Fc is present). In preferred embodiments, the trivalent bispecific antibody has the format BCMA Fab - Fc - CD3 Fab - BCMA Fab.

In some embodiments, the anti-CD3 Fab comprises a light chain and a heavy chain, wherein the light chain is a crossover light chain that comprises a variable domain VH and a constant domain CF, and wherein the heavy chain is a crossover heavy chain that comprises a variable domain VF and a constant domain CHI .

In some embodiments, the CHI domain of the anti-BCMA Fab fragment comprises the amino acid modifications K147E/D and K213E/D (numbered according to EU numbering) and a corresponding immunoglobulin light chain comprising a CF domain having amino acid modifications E123K/R/H and Q124K/R/H (numbered according to Kabat).

In some embodiments, the multispecific (e.g. bispecific) antibody further comprises an Fc. In some embodiments, the Fc is an IgGl Fc. In some embodiments, the (e.g. IgGl) Fc comprises a first Fc chain comprising first constant domains CH2 and CH3, and a second Fc chain comprising second constant domains CH2 and CH3, and wherein: a) the first CH3 domain comprises the modifications T366S, F368A and Y407V, or conservative substitutions thereof (numbered according to EU numbering); and b) the second CH3 domain comprises the modifications T366W, or conservative substitutions thereof (numbered according to EU numbering). In some embodiments, the (e.g. IgGl) Fc comprises: a) the modifications L234A, L235A and P329G (numbered according to EU numbering); and/or b) the modifications D356E, and L358M (numbered according to EU numbering).

In further embodiments, the bispecific antibody according to the invention comprises the following SEQ ID NOs: l. 83A10-TCBcv: 45, 46, 47 (x2), 48 (Figure 2A) li. 21-TCBcv: 48, 49, 50, 51 (x2) (Figure 2A) in. 22-TCBcv: 48, 52, 53, 54 (x2) (Figure 2A) lv. 42-TCBcv: 48, 55, 56, 57 (x2) (Figure 2A).

In a preferred embodiment, the bispecific antibody according to the invention is 42-TCBcv.

Aspects and embodiments of the invention are set out in the appended claims. These and other aspects and embodiments of the invention are also described herein.

BRIEF DESCRIPTION OF FIGURES

The present invention will now be described in more detail with reference to the attached Figures, in which:

Figure 1 illustrates different formats of bispecific bivalent antibodies for use in the present invention, which comprise Fab fragments binding to a T cell antigen (CD3 is illustrated) and BCMA in the format Fab BCMA- Fc - Fab CD3. The CD3 Fab may include a VH-VL crossover to reduce light chain mispairing and side-products. Amino acid substitutions “RK/EE” may be introduced in CL-CH1 to reduce light chain mispairing/side products in production. The CD3 Fab and BCMA Fab may be linked to each other with flexible linkers.

Figure 2 illustrates different formats of bispecific trivalent antibodies for use in the present invention, which comprise Fab fragments binding to a T cell antigen (CD3 is illustrated) and BCMA in the following formats: Fab BCMA - Fc - Fab CD3 - Fab BCMA (A,B); Fab BCMA - Fc - Fab BCMA - Fab CD3 (C,D). The CD3 Fab may include a VH-VL crossover to reduce light chain mispairing and side-products. Amino acid substitutions “RK/EE” may be introduced in CL-CH1 to reduce light chain mispairing/side products in production. The CD3 Fab and BCMA Fab may be linked to each other with flexible linkers.

Figure 3 illustrates further formats of bispecific bivalent antibodies for use in the present invention, which comprise Fab fragments binding to a T cell antigen (CD3 is illustrated) and BCMA in the following formats: Fc - Fab CD3 - Fab BCMA (A,B); Fc -Fab BCMA - Fab CD3 (C,D). The CD3 Fab may include a VH-VL crossover to reduce light chain mispairing and side-products. Amino acid substitutions “RK/EE” may be introduced in CL-CH1 to reduce light chain mispairing/side products in production. The CD3 Fab and BCMA Fab may be linked to each other with flexible linkers

Figure 4 illustrates BCMA cell surface expression in 37 lymphoma cell lines from a range of cellular origins. An isogenic pair of K562 that is null for BCMA expression and K562 constitutively overexpressing BCMA (K562-BCMA) served as negative and positive control cell lines, respectively. The multiple myeloma (MM) cell line H929 was also profiled. BCMA surface expression varies among NHL cell lines, ranging from 43 to 17,048 molecules per cell (median, 420). BCMA surface expression is much lower than the MM cell line for nearly all of the tested lymphoma cell lines.

Figure 5 illustrates the correlation between BCMA cell surface expression (mean fluorescent intensity, MFI) and BCMA mRNA expression (r 2 , 0.3377; p<0.001).

Figure 6 illustrates T-cell mediated cytotoxicity against 10 of 11 NHL cell lines treated with CC- 93269 for 72 hours (IC50 range from 0.1 to 38 ng/ml), even in cells with very low BCMA cell surface expression.

Figure 7 illustrates representative immunohistochemistry analysis of BCMA cell surface expression in DLBCL and FL tumors using an anti-BCMA antibody.

Figure 8 illustrates immunohistochemistry analysis of frontline DLBCL biopsies, R/R DLBCL biopsies and frontline FL biopsies demonstrating a range of BCMA cell surface expression levels and intensities amongst the malignant B cells (Pax5+).

Figure 9 illustrates scatterplots to visualize the percentage of BCMA-positive tumor cells with staining intensity in B-NHL tumor sections.

Figure 10 illustrates the percentage of tumor sections which are considered to be BCMA-expressing by IHC, using different thresholds for the percentage of BCMA-positive tumor cells.

Figure 11 illustrates the effect of CC-93269 on FL tumor samples. FL were treated for 7 days with CC-93269 (“BCMA”) at a concentration of 1 pg/ml. Vehicle and a CD20xCD3 bispecific antibody (“TCB”) served as negative and positive controls, respectively. Shown are the B cell (A) and T cell (B) count on FL samples following treatment.

DETAILED DESCRIPTION

As used herein, the articles "a" and “an” may refer to one or to more than one (e.g. to at least one) of the grammatical object of the article.

“About” may generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Exemplary degrees of error are within 20 percent (%), typically, within 10%, and more typically, within 5% of a given value or range of values.

Embodiments described herein as “comprising” one or more features may also be considered as disclosure of the corresponding embodiments “consisting of’ and/or “consisting essentially of’ such features.

Concentrations, amounts, volumes, percentages and other numerical values may be presented herein in a range format. It is also to be understood that such range format is used merely for convenience and brevity and should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited.

Therapeutic methods

The invention is based, at least in part, on the treatment of a patient having a lymphoma (e.g. non- Hodgkin’s Lymphoma (NHL)) with multispecific (e.g. bispecific) antibodies that bind to an antigen that promotes activation of one or more T cells (e.g. CD3) and BCMA. In certain embodiments, the “subject” or “patient” is a human.

As used herein, the terms “treat,” “treating” or “treatment,” and the like refer to obtaining a desired pharmacologic and/or physiologic effect. Preferably, the effect is therapeutic, i.e., the effect partially or completely cures a disease and/or adverse symptom attributable to the disease. Alternatively, the pharmacologic and/or physiologic effect may be prophylactic, i.e., the effect completely or partially prevents a disease or symptom thereof.

The present invention relates to the treatment of lymphoma with multispecific (e.g. bispecific) antibodies that bind to an antigen that promotes activation of one or more T cells (e.g. CD3) and BCMA.

In some embodiments, the lymphoma has BCMA cell surface expression of at least about 40 molecules per cell, at least about 50 molecules per cell, at least about 60 molecules per cell, at least about 100 molecules per cell, or at least about 250 molecules per cell, as measured using a flow cytometry system based on a logarithmic dilution of phycoerythrin beads of a known quantity. In some embodiments, the lymphoma has BCMA cell surface expression of at least 45 molecules per cell, as measured using a flow cytometry system based on a logarithmic dilution of phycoerythrin beads of a known quantity.

In some embodiments, the lymphoma has BCMA cell surface expression of less than BCMA cell surface expression on a multiple myeloma cell (e.g. H929 cell line). In some embodiments, the lymphoma has BCMA cell surface expression of less than about 7000 molecules per cell, less than about 6000 molecules per cell, less than about 5000 molecules per cell, less than about 4000 molecules per cell, as measured using a flow cytometry system based on a logarithmic dilution of phycoerythrin beads of a known quantity.

In some embodiments, the lymphoma has BCMA cell surface expression of between about 45 and about 17,500 molecules per cell, between about 45 and about 10,000 molecules per cell, between about 45 and about 7500 molecules per cell, between about 45 and about 6000 molecules per cell, between about 45 and about 5000 molecules per cell, between about 45 and about 4000 molecules per cell, between about 45 and about 3000 molecules per cell, between about 45 and about 2000 molecules per cell, between about 45 and about 1000 molecules per cell, as measured using a flow cytometry system based on a logarithmic dilution of phycoerythrin beads of a known quantity.

In some embodiments, the lymphoma is a Hodgkin’s lymphoma (HL) or a non-Hodgkin’s lymphoma (NHL). In preferred embodiments, the lymphoma is a NHL.

The majority of cases of NHL and HL are B cell lymphomas, i.e. lymphomas that originate in B cells. In preferred embodiments, the lymphoma, e.g. HL or NHL, is a B cell lymphoma. In particularly preferred embodiments, the lymphoma is a B cell NHL.

B cell lymphomas can arise from germinal center B cells or activated B cells. The multispecific (e.g. bispecific) antibodies of the invention may treat B cell lymphoma regardless of the B cell of origin, e.g. the multispecific (e.g. bispecific) antibodies of the invention may treat activated B cell lymphomas and germinal center B cell lymphomas. In some embodiments, the multispecific (e.g. bispecific) antibodies of the invention treat activated B cell lymphomas. In some embodiments, the multispecific (e.g. bispecific) antibodies of the invention treat germinal center B cell lymphomas.

In some embodiments, the multispecific (e.g. bispecific) antibodies of the invention treat diffuse large B-cell lymphoma (DLBCL). DLBCL is the most common subtype of NHL and is the most prevalent type of aggressive lymphoma. In some embodiments, the multispecific (e.g. bispecific) antibodies of the invention treat follicular lymphoma. Follicular lymphoma is the largest constituent of indolent lymphoma. In some embodiments, the multispecific (e.g. bispecific) antibodies of the invention treat mantle cell lymphoma. In some embodiments, the multispecific (e.g. bispecific) antibodies of the invention treat diffuse large B-cell lymphoma (DLBCL), follicular lymphoma and mantle cell lymphoma.

In some embodiments, the lymphoma (e.g. NHL) is relapsed or refractory lymphoma (e.g. relapsed or refractory NHL). As used herein, the term “relapsed” is intended to mean the return of the cancer or the signs and symptoms of the cancer after a period of improvement. As used herein, the term “refractory” is intended to mean that the particular cancer is resistant to, or non-responsive to, therapy with a particular therapeutic agent. A cancer can be refractory to therapy with a particular therapeutic agent either from the onset of treatment with the particular therapeutic agent (i.e., non- responsive to initial exposure to the therapeutic agent), or as a result of developing resistance to the therapeutic agent, either over the course of a first treatment period with the therapeutic agent or during a subsequent treatment period with the therapeutic agent.

Approximately one-third of patients with DLBCL have refractory disease or experience relapse after the standard therapies. In some embodiments, the multispecific (e.g. bispecific) antibodies of the invention treat front line DLBCL. In some embodiments, the multispecific (e.g. bispecific) antibodies of the invention treat relapsed or refractory DLBCL. In particularly preferred embodiments, the multispecific (e.g. bispecific) antibodies of the invention treat frontline DLBCL and relapsed or refractory DLBCL. In preferred embodiments, the lymphoma is selected from the group consisting of: relapsed or refractory DLBCL; relapsed or refractory follicular lymphoma; relapsed or refractory mantle cell lymphoma; and combinations thereof.

Lymphomas amenable to treatment with a multispecific (e.g. bispecific) antibodies of the invention include, without limitation, non-Hodgkin’s lymphoma (NHL), including diffuse large B cell lymphoma (DLBCL), which may be relapsed or refractory DLBCL, as well as other lymphomas including germinal-center B cell-like (GCB) diffuse large B cell lymphoma (DLBCL), activated B cell-like (ABC) DLBCL, follicular lymphoma (FL), mantle cell lymphoma (MCL), acute myeloid leukemia (AML), chronic lymphoid leukemia (CLL), small lymphocytic leukemia (SLL), marginal zone lymphoma (MZL), lymphoplasmacytic lymphoma (LL), Waldenstrom macroglobulinemia (WM), central nervous system lymphoma (CNSL), Burkitt’s lymphoma (BL), splenic marginal zone lymphoma, splenic lymphoma/leukemia, unclassifiable, splenic diffuse red pulp small B cell lymphoma, extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma), non-Burkitt high grade B cell lymphoma, nodal marginal zone lymphoma, primary pediatric nodal marginal zone lymphoma, pediatric follicular lymphoma, primary cutaneous follicle center lymphoma, intraocular lymphoma, T cell/histiocyte rich large B cell lymphoma, primary DLBCL of the CNS, primary cutaneous DLBCL, leg type, EBV-positive DLBCL of the elderly, DLBCL associated with chronic inflammation, lymphomatoid granulomatosis, primary mediastinal (thymic) large B cell lymphoma (PMLBCL), intravascular large B cell lymphoma, ALK-positive large B cell lymphoma, plasmablastic lymphoma, large B cell lymphoma arising in HHV8- associated multicentric Castleman disease, primary effusion lymphoma: B cell lymphoma, unclassifiable, with features intermediate between DLBCL and Burkitt lymphoma, and B cell lymphoma, unclassifiable, with features intermediate between DLBCL and classical Hodgkin’s lymphoma. Further examples of lymphoma include, but are not limited to, low grade/follicular NHL; small lymphocytic (SL) NHL; intermediate grade/follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non- cleaved cell NHL; bulky disease NHL; AIDS-related lymphoma; and post-transplant lymphoproliferative disorder (PTLD). In preferred embodiments, the lymphoma is DLBCL (e.g., relapsed or refractory DLBCL) or follicular lymphoma.

T cell directed tumor lysis

The multispecific, e.g. bispecific, antibodies of the invention bind to an antigen that promotes activation of one or more T cells, e.g. a T cell antigen. As used herein, the term “T cell antigen” refers to an antigen that promotes activation of one or more T cells.

In preferred embodiments, the T cell antigen is a human T cell antigen. Thus, binding of the multispecific, e.g. bispecific, antibodies of the invention to a T cell antigen may allow for recruitment of the T cell to the BCMA-expressing lymphoma cell to result in lysis of the lymphoma cell. The multispecific, e.g. bispecific, antibodies of the invention are therefore capable of inducing tumor killing by redirecting cytotoxic T cells to the BCMA-expressing lymphoma cells. In preferred embodiments, the T cell antigen is CD3.

Lymphoma (e.g. NHL) cells may be incubated with multispecific, e.g. bispecific, antibodies of the invention and isolated CD3+ T-cells at 1:5 lymphoma cell to effector cell ratio for 72 hours, prior to the assessment of cell lysis. In some embodiments, lymphoma cell lysis is quantified by measured by annexin V and ToPro-3 staining of CFSE-positive lymphoma cells.

The multispecific, e.g. bispecific, antibodies of the invention which bind to BCMA and a T cell antigen, preferably CD3, may achieve lysis at a concentration of 200 ng/ml, 100 ng/ml or 50 ng/ml of the BCMA-expressing lymphoma cell (e.g. JEKO-1, Farage, TMD8, OCI-LY10, U2932, Riva, SUDHL-4, SUDHL- 10, SUDHL- 16, WSU DLCL2, Karpas 422 or Pfeiffer) of more than more than 40%, more than 50%, more than 60%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 95% or about 100%, as compared to a non-BCMA-expressing control, in the above-described cell lysis assay. In embodiments in which the BCMA-expressing cell line is JEKO-1, OCI-LY10, U2932, Riva, WSU DLCL2, SUDHL-4, SUDHL-10, TMD8, the multispecific, e.g. bispecific, antibodies of the invention which bind to BCMA and a T cell antigen, preferably CD3, may achieve lysis at a concentration of 200 ng/ml, 100 ng/ml or 50 ng/ml of more than 80%, more than 85%, more than 90%, more than 95% or about 100%, as compared to a non- BCMA-expressing control, in the above-described cell lysis assay.

Adverse events

In some embodiments, the patient develops, or is at risk of developing, an adverse event associated with the administration of the multispecific (e.g. bispecific) antibody. The adverse event may be cytokine-driven toxicities (e.g. cytokine release syndrome (CRS)), infusion-related reactions (IRRs), macrophage activation syndrome (MAS), neurologic toxicities, severe tumor lysis syndrome (TLS), neutropenia, thrombocytopenia, elevated liver enzymes, and/or central nervous system (CNS) toxicities. In particular embodiments, the adverse event is CRS. In the event that the patient develops, or is at risk of developing, an adverse event associated with the administration of the multispecific (e.g. bispecific) antibody, the treatment further comprises the administration of an agent capable of treating, preventing, delaying, reducing or attenuating the development or risk of development of the adverse event. The agent may be administered to the patient prior to the initiation of the treatment with the multispecific (e.g. bispecific) antibody (e.g. as a prophylaxis in order to prevent or reduce the risk of an adverse event developing) or during treatment with the multispecific (e.g. bispecific) antibody (e.g. in response to the development of an adverse event).

In some embodiments, the agent comprises a steroid, such as a corticosteroid. As used herein, "corticosteroid" means any naturally occurring or synthetic steroid hormone that can be derived from cholesterol and is characterized by a hydrogenated cyclopentanoperhydrophenanthrene ring system. Naturally occurring corticosteroids are generally produced by the adrenal cortex. Synthetic corticosteroids may be halogenated. Functional groups required for activity include a double bond at D4, a C3 ketone, and a C20 ketone. Corticosteroids may have glucocorticoid and/or mineralocorticoid activity. Examples of exemplary corticosteroids include prednisolone, methylprednisolone, prednisone, triamcinolone, betamethasone, budesonide, and dexamethasone.

In some embodiments, the agent comprises an antagonist of a cytokine receptor or cytokine selected from among GM-CSF, IL-10, IL-10R, IL-6, IL-6 receptor (IL-6R), IFNy, IFNGR, IL-2, IL- 2R/CD25, MCP-1, CCR2, CCR4, MIRIb, CCR5, TNFalpha, TNFR1, IL-1, and IL-lRalpha/IL- lbeta, wherein the antagonist is selected from an antibody or antigen-binding fragment, a small molecule, a protein or peptide and a nucleic acid. The antagonist may be an anti-IL-6 antibody and/or an anti-IL6R antibody. For example, the antagonist may be selected from tocilizumab, siltuximab, clazakizumab, sarilumab, olokizumab, elsilimomab, ALD518/BMS-945429, sirukumab (CNTO 136), CPSI-2634, ARGX-109, lenzilumab, FE301 and FM101. In some embodiments, the antagonist is tocilizumab and/or siltuximab.

In some embodiments, the agent comprises a molecule that decreases the regulatory T cell (Treg) population. Agents that decrease the number of (e.g., deplete) Treg cells are known in the art and include, e.g., CD25 depletion, cyclophosphamide administration, anti-CTLA4 antibody and modulating Glucocorticoid-induced TNLR family related gene (GITR) function. GITR is a member of the TNLR superfamily that is upregulated on activated T cells, which enhances the immune system. In some embodiments, the treatment comprises the administration of cyclophosphamide. The multispecific antibody

The multispecific (e.g. bispecific) antibodies of the invention specifically bind to BCMA and to an antigen that promotes activation of one or more T cells (e.g. CD3). The terms “antibody against BCMA and an antigen that promotes activation of one or more T cells (e.g. CD3)”, or “an antibody that binds to BCMA and to an antigen that promotes activation of one or more T cells (e.g. CD3),” refer to a multispecific antibody (e.g., a bispecific antibody) that is capable of binding to BCMA and an antigen that promotes activation of one or more T cells (e.g. CD3) with sufficient affinity such that the antibody is useful as a therapeutic agent. This is achieved by making a molecule which comprises a first antibody, or antigen-binding fragment, that binds to BCMA and a second antibody, or antigen-binding fragment, that binds to an antigen that promotes activation of one or more T cells (e.g. CD3). Such multispecific antibodies may be trispecific antibodies or bispecific antibodies hi preferred embodiments, the multispecific antibodies are bispecific antibodies.

The term “BCMA” as used herein relate to human B cell maturation antigen, also known as BCMA; TR17_HUMAN, TNFRSF17 (UniProt Q02223), which is a member of the tumor necrosis receptor superfamily that is preferentially expressed in differentiated plasma cells. The extracellular domain of BCMA consists according to UniProt of amino acids 1-54 (or 5-51). The terms “antibody against BCMA”, “anti BCMA antibody” or “an antibody that binds to BCMA” as used herein relate to an antibody specifically binding to the extracellular domain of BCMA.

The term “specifically binding to BCMA” refers to an antibody that is capable of binding to the defined target with sufficient affinity such that the antibody is useful as a therapeutic agent in targeting BCMA. In some embodiments, an antibody specifically binding to BCMA does not bind to other antigens, or does not bind to other antigens with sufficient affinity to produce a physiological effect.

In some embodiments, the extent of binding of an anti-BCMA antibody to an unrelated, non-BCMA protein is about 10-fold preferably >100-fold less than the binding of the antibody to BCMA as measured, e.g., by surface plasmon resonance (SPR) e.g. Biacore®, enzyme-linked immunosorbent (ELISA) or flow cytometry (FACS). In one embodiment the antibody that binds to BCMA has a dissociation constant (Kd) of 10 8 M or less, preferably from 10 8 M to 10 13 M, preferably from 10 9 Mto 10 13 M.

In one embodiment the anti-BCMA antibody binds to an epitope of BCMA that is conserved among BCMA from different species, preferably among human and cynomolgus, and in addition preferably also to mouse and rat BCMA.

Preferably the anti -BCMA antibody specifically binds to a group of BCMA, consisting of human BCMA and BCMA of non-human mammalian origin, preferably BCMA from cynomolgus, mouse and/or rat. Anti -BCMA antibodies are analyzed by ELISA for binding to human BCMA using plate- bound BCMA. For this assay, an amount of plate-bound BCMA preferably 1.5 pg/mL and concentration(s) ranging from 0.1 pM to 200 nM of anti-BCMA antibody are used.

The multispecific (e.g. bispecific) antibodies of the invention bind to an antigen that promotes activation of one or more T cells, e.g. a T cell antigen. In preferred embodiments, the T cell antigen is a human T cell antigen. The antigen that promotes activation of one or more T cells, e.g. T cell antigen, may be selected from the group consisting of CD3, TCRa, TCR-b, TCRy, TCR-z, ICOS, CD28, CD27, HVEM, LIGHT, CD40, 4-1BB, 0X40, DR3, GITR, CD30, TIM1, SLAM, CD2, or CD226. In preferred embodiments, the antigen that promotes activation of one or more T cells, e.g. T cell , is CD3, e.g. human CD3. Accordingly, in preferred embodiments, multispecific (e.g. bispecific) antibodies of the invention bind to CD3, e.g. human CD3.

The term “CD3” refers to the human CD3 protein multi-subunit complex. The CD3 protein multi subunit complex is composed to 6 distinctive polypeptide chains. Thus the term includes a CD3y chain (SwissProt P09693), a CD35 chain (SwissProt P04234), two CD3s chains (SwissProt P07766), and one Oϋ3z chain homodimer (SwissProt 20963), and which is associated with the T cell receptor a and b chain. The term encompasses “full-length,” unprocessed CD3, as well as any CD3 variant, isoform and species homolog which is naturally expressed by cells (including T cells) or can be expressed on cells transfected with genes or cDNA encoding those polypeptides.

The term “specifically binding to CD3” refers to an antibody that is capable of binding to the defined target with sufficient affinity such that the antibody is useful as a therapeutic agent in targeting CD3. In some embodiments, an antibody specifically binding to CD3 does not bind to other antigens, or does not bind to other antigens with sufficient affinity to produce a physiological effect.

The multispecific (e.g. bispecific) antibodies of the invention can be analysed by SPR, e.g. Biacore®, for binding to CD3. In some embodiments, the bispecific antibodies bind to human CD3 with a dissociation constant (K D ) of about 10 7 M or less, a K D of about lO 8 M or less, a K D of about 10 9 M or less, a K D of about lO 10 M or less, a K D of about lO 11 M or less, or a K D of about lO 12 M or less, as determined by a surface plasmon resonance assay, preferably measured using Biacore 8K at 25°C. In preferred embodiments, the bispecific antibodies bind to human CD3 with a dissociation constant (K D ) of about 10 8 M or less.

The term “antibody” herein encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity.

A “heavy chain” comprises a heavy chain variable region (abbreviated herein as “VH”) and a heavy chain constant region (abbreviated herein as “CH”). The heavy chain constant region comprises the heavy chain constant domains CHI, CH2 and CH3 (antibody classes IgA, IgD, and IgG) and optionally the heavy chain constant domain CH4 (antibody classes IgE and IgM).

A “light chain” comprises a light chain variable domain (abbreviated herein as “VL”) and a light chain constant domain (abbreviated herein as “CL”). The variable regions VH and VL can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is 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. The “constant domains” of the heavy chain and of the light chain are not involved directly in binding of an antibody to a target, but exhibit various effector functions.

Binding between an antibody and its target antigen or epitope is mediated by the Complementarity Determining Regions (CDRs). The CDRs are regions of high sequence variability, located within the variable region of the antibody heavy chain and light chain, where they form the antigen-binding site. The CDRs are the main determinants of antigen specificity. Typically, the antibody heavy chain and light chain each comprise three CDRs which are arranged non-consecutively. The antibody heavy and light chain CDR3 regions play a particularly important role in the binding specificity/affmity of the antibodies according to the invention and therefore provide a further aspect of the invention.

The term “antigen binding fragment” as used herein incudes any naturally-occurring or artificially- constructed configuration of an antigen-binding polypeptide comprising one, two or three light chain CDRs, and/or one, two or three heavy chain CDRs, wherein the polypeptide is capable of binding to the antigen. Thus, the term refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include but are not limited to Fv, Fab, Fab’, Fab’-SH, F(ab’)2; diabodies; linear antibodies; single-chain antibody molecules (e.g. scFv); and multispecific antibodies formed from antibody fragments.

The terms “Fab fragment” and “Fab” are used interchangeably herein and contain a single light chain (i.e. a constant domain CL and a VL) and a single heavy chain (i.e. the constant domain CHI and a VH). The heavy chain of a Fab fragment is not capable of forming a disulfide bond with another heavy chain.

A “Fab 1 fragment” contains a single light chain and a single heavy chain but in addition to the CHI and the VH, a “Fab 1 fragment” contains the region of the heavy chain between the CHI and CH2 domains that is required for the formation of an inter-chain disulfide bond. Thus, two “Fab 1 fragments” can associate via the formation of a disulphide bond to form a F(ab')2 molecule.

A “F(ab')2 fragment” contains two light chains and two heavy chains. Each chain includes a portion of the constant region necessary for the formation of an inter-chain disulfide bond between two heavy chains.

An “Fv fragment” contains only the variable regions of the heavy and light chain. It contains no constant regions.

A “single-domain antibody” is an antibody fragment containing a single antibody domain unit (e.g., VH or VL).

A “single-chain Fv” (“scFv”) is antibody fragment containing the VH and VL domain of an antibody, linked together to form a single chain. A polypeptide linker is commonly used to connect the VH and VL domains of the scFv.

A “tandem scFv”, also known as a TandAb ® , is a single-chain Fv molecule formed by covalent bonding of two scFvs in a tandem orientation with a flexible peptide linker.

A “bi-specific T cell engager” (BiTE ® ) is a fusion protein consisting of two single-chain variable fragments (scFvs) on a single peptide chain. One of the scFvs binds to T cells via the CD3 receptor, and the other to a tumor cell antigen.

A “diabody” is a small bivalent and bispecific antibody fragment comprising a heavy (VH) chain variable domain connected to a light chain variable domain (VL) on the same polypeptide chain (VH-VL) connected by a peptide linker that is too short to allow pairing between the two domains on the same chain (Kipriyanov, Int. J. Cancer 77 (1998), 763-772). This forces pairing with the complementary domains of another chain and promotes the assembly of a dimeric molecule with two functional antigen binding sites.

A “DARPin” is a bispecific ankyrin repeat molecule. DARPins are derived from natural ankyrin proteins, which can be found in the human genome and are one of the most abundant types of binding proteins. A DARPin library module is defined by natural ankyrin repeat protein sequences, using 229 ankyrin repeats for the initial design and another 2200 for subsequent refinement. The modules serve as building blocks for the DARPin libraries. The library modules resemble human genome sequences. A DARPin is composed of 4 to 6 modules. Because each module is approx. 3.5 kDa, the size of an average DARPin is 16-21 kDa. Selection of binders is done by ribosome display, which is completely cell-free and is described in He M. and Taussig MX, Biochem Soc Trans. 2007, Nov; 35 (R 5):962-5.

The sequence of a CDR may be identified by reference to any number system known in the art, for example, the Kabat system (Kabat, E. A., et al, Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991); the Chothia system (Chothia &, Lesk, “Canonical Structures for the Hypervariable Regions of Immunoglobulins,” J. Mol. Biol. 196, 901-917 (1987)); or the IMGT system (Lefranc et al., “IMGT Unique Numbering for Immunoglobulin and Cell Receptor Variable Domains and Ig superfamily V-like domains,” Dev. Comp. Immunol. 27, 55-77 (2003)).

Table 1: CDR definitions For heavy chain constant region amino acid positions discussed in the invention, numbering is according to the EU index first described in Edelman, G.M., et al., Proc. Natl. Acad. Sci. USA 63 (1969) 78-85). The EU numbering of Edelman is also set forth in Kabat et al. (1991) {supra.). Thus, the terms “EU index as set forth in Kabat”, “EU Index”. “EU index of Kabat” or “EU numbering” in the context of the heavy chain refers to the residue numbering system based on the human lgGl EU antibody of Edelman et al. as set forth in Kabat et al. (1991). The numbering system used for the light chain constant region amino acid sequence is similarly set forth in Kabat et al. (supra.). Thus, as used herein, “numbered according to Kabat” refers to the Kabat set forth in Kabat et al. {supra ).

The antibodies of the invention and antigen-binding fragments thereof may be derived from any species by recombinant means. For example, the antibodies or antigen-binding fragments may be mouse, rat, goat, horse, swine, bovine, chicken, rabbit, camelid, donkey, human, or chimeric versions thereof. For use in administration to humans, non-human derived antibodies or antigen binding fragments may be genetically or structurally altered to be less antigenic upon administration to the human patient.

Especially preferred are human or humanized antibodies, especially as recombinant human or humanized antibodies.

The term “humanized antibody” refers to antibodies in which the framework or “complementarity determining regions” (CDRs) have been modified to comprise the CDR of an immunoglobulin of different specificity as compared to that of the parent immunoglobulin. For example, a murine CDR may be grafted into the framework region of a human antibody to prepare the “humanized antibody.” See, e.g., Riechmann, L., et al., Nature 332 (1988) 323-327; and Neuberger, M.S., et al, Nature 314 (1985) 268-270. In some embodiments, “humanized antibodies” are those in which the constant region has been additionally modified or changed from that of the original antibody to generate the properties of the antibodies according to the invention, especially in regard to Clq binding and/or Fc receptor (FcR) binding.

The term “human antibody” is one which possesses an amino acid sequence which corresponds to that of an antibody produced by a human or a human cell or derived from a non-human source that utilizes human antibody repertoires or other human antibody-encoding sequences. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen binding residues. Human antibodies can be produced using various techniques known in the art, including phage-display libraries. The term “chimeric antibody” refers to an antibody comprising a variable region, i.e., binding region, from one source or species and at least a portion of a constant region derived from a different source or species, usually prepared by recombinant DNA techniques. Chimeric antibodies comprising a murine variable region and a human constant region are preferred. Other preferred forms of “chimeric antibodies” encompassed by the present invention are those in which the constant region has been modified or changed from that of the original antibody to generate the properties of the antibodies according to the invention, especially in regard to Clq binding and/or Fc receptor (FcR) binding. Such chimeric antibodies are also referred to as “class-switched antibodies”. Chimeric antibodies are the product of expressed immunoglobulin genes comprising DNA segments encoding immunoglobulin variable regions and DNA segments encoding immunoglobulin constant regions. Methods for producing chimeric antibodies involving conventional recombinant DNA and gene transfection techniques are well known in the art. See, e.g., Morrison, S.L., et al, Proc. Natl. Acad. Sci. USA 81 (1984) 6851-6855; US Patent Nos. 5,202,238 and 5,204,244.

The terms “Fc region” and “Fc” are used interchangeably herein and refer to the portion of a native immunoglobulin that is formed by two Fc chains. Each “Fc chain” comprises a constant domain CH2 and a constant domain CH3. Each Fc chain may also comprise a hinge region. A native Fc region is homodimeric. In some embodiments, the Fc region may contain modifications to enforce Fc heterodimerization.

The term “Fc part” refers to the portion of an antibody of the invention, or antigen binding fragment thereof, which corresponds to the Fc region.

There are five major classes of heavy chain constant region, classified as IgA, IgG, IgD, IgE and IgM, each with characteristic effector functions designated by isotype. For example, IgG is separated into four subclasses known as IgGl, IgG2, IgG3, and IgG4. Ig molecules interact with multiple classes of cellular receptors. For example, IgG molecules interact with three classes of Fey receptors (FcyR) specific for the IgG class of antibody, namely FcyRI, FcyRII, and FcyRIII. The important sequences for the binding of IgG to the FcyR receptors have been reported to be located in the CH2 and CH3 domains.

The antibodies of the invention or antigen-binding fragments thereof may be any isotype, i.e. IgA, IgD, IgE, IgG and IgM, and synthetic multimers of the four-chain immunoglobulin (Ig) structure. In preferred embodiments, the antibodies or antigen-binding fragments thereof are IgG isotype. The antibodies or antigen-binding fragments can be any IgG subclass, for example IgGl, IgG2, IgG3, or IgG4 isotype. In preferred embodiments, the antibodies or antigen-binding fragments thereof are of an IgGl isotype.

In some embodiments, the antibodies comprise a heavy chain constant region that is of IgG isotype. In some embodiments, the antibodies comprise a portion of a heavy chain constant region that is of IgG isotype. In some embodiments, the IgG constant region or portion thereof is an IgGl, IgG2, IgG3, or IgG4 constant region. Preferably, the IgG constant region or portion thereof is an IgGl constant region.

The antibodies of the invention or antigen-binding fragments thereof may comprise a lambda light chain or a kappa light chain.

In preferred embodiments, the antibodies or antigen-binding fragments thereof comprise a light chain that is a kappa light chain. In some embodiments, the antibody or antigen-binding fragment comprises a light chain comprising a light chain constant region (CL) that is a kappa constant region.

In some embodiments, the antibody comprises a light chain comprising a light chain variable region (VL) that is a kappa variable region. Preferably, the kappa light chain comprises a VL that is a kappa VL and a CL that is a kappa CL.

Alternatively, the antibodies or antigen-binding fragments thereof may comprise a light chain that is a lambda light chain. In some embodiments, the antibody or antigen-binding fragment comprises a light chain comprising a light chain constant region (CL) that is a lambda constant region. In some embodiments, the antibody comprises a light chain comprising a light chain variable region (VL) that is a lambda variable region.

Engineered antibodies and antigen-binding fragments thereof include those in which modifications have been made to framework residues within the VH and/or VL. Such modifications may improve the properties of the antibody, for example to decrease the immunogenicity of the antibody and/or improve antibody production and purification.

Antibodies and antigen-binding fragments thereof disclosed herein can be further modified using conventional techniques known in the art, for example, by using amino acid deletion(s), insertion(s), substitution(s), addition(s), and/or recombination(s) and/or any other modification(s) known in the art, either alone or in combination. Methods for introducing such modifications in the DNA sequence underlying the amino acid sequence of an immunoglobulin chain arc well known to the person skilled in the art.

The antibodies of the invention and antigen-binding fragments thereof also include derivatives that are modified ( e.g ., by the covalent attachment of any type of molecule to the antibody) such that covalent attachment does not prevent the antibody from binding to its epitope, or otherwise impair the biological activity of the antibody. Examples of suitable derivatives include, but are not limited to fucosylated antibodies, glycosylated antibodies, acetylated antibodies, PEGylated antibodies, phosphorylated antibodies, and amidated antibodies.

Minor variations in the amino acid sequences of antibodies of the invention are contemplated as being encompassed by the present invention, providing that the variations in the amino acid sequence(s) maintain at least 75%, more preferably at least 80%, at least 90%, at least 95%, and most preferably at least 99% sequence identity to the antibody of the invention or antigen-binding fragment thereof as defined anywhere herein.

Antibodies of the invention may include variants in which amino acid residues from one species are substituted for the corresponding residue in another species, either at the conserved or non- conserved positions. In one embodiment, amino acid residues at non-conserved positions are substituted with conservative or non-conservative residues. In particular, conservative amino acid replacements are contemplated.

A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, or histidine), acidic side chains (e.g., aspartic acid or glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, or cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, or tryptophan), beta- branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, or histidine). Thus, if an amino acid in a polypeptide is replaced with another amino acid from the same side chain family, the amino acid substitution is considered to be conservative. The inclusion of conservatively modified variants in an antibody of the invention does not exclude other forms of variant, for example polymorphic variants, interspecies homologs, and alleles.

“Non-conservative amino acid substitutions” include those in which (i) a residue having an electropositive side chain (e.g., Arg, His or Lys) is substituted for, or by, an electronegative residue (e.g., Glu or Asp), (ii) a hydrophilic residue (e.g., Ser or Thr) is substituted for, or by, a hydrophobic residue (e.g., Ala, Leu, lie, Phe or Val), (iii) a cysteine or proline is substituted for, or by, any other residue, or (iv) a residue having a bulky hydrophobic or aromatic side chain (e.g., Val, His, lie or Trp) is substituted for, or by, one having a smaller side chain (e.g., Ala or Ser) or no side chain (e.g., Gly).

Antibody format

Formats for multispecific, e.g. bispecific, antibodies are known in the state of the art. For example, bispecific antibody formats are described in Kontermann RE, mAbs 4:2 1-16 (2012); Holbger P., Hudson PJ, Nature Biotech.23 (2005) 1126- 1136, Chan AC, Carter PJ Nature Reviews Immunology 10, 301-316 (2010) and Cuesta AM et al., Trends Biotech 28 (2011) 355-362.

The multispecific, e.g. bispecific, antibodies of the invention may have any format. Multispecific and bispecific antibody formats include, for example, multivalent single chain antibodies, diabodies and triabodies, and antibodies having the constant domain structure of full length antibodies to which further antigen-binding domains (e.g., single chain Fv, a tandem scFv, a VH domain and/or a VL domain, Fab, or (Fab)2,) are linked via one or more peptide-linkers, as well as antibody mimetics such as DARPins. In some embodiments, the multispecific, e.g. bispecific, antibodies of the invention have the format of an scFv such as a bispecific T cell engager (BITE ® ). In some embodiments, the antibodies of the invention are single chain antibodies which comprise a first domain which binds to BCMA, a second domain which binds to a T cell antigen (e.g. CD3), and a third domain which comprises two polypeptide monomers, each comprising a hinge, a CH2 domain and a CH3 domain, wherein the two polypeptide monomers are fused to each other via a peptide linker (e.g. (hinge-CH2-CH3-linker-hinge-CH2-CH3).

The “valency” of an antibody denotes the number of binding domains. As such, the terms "bivalent", “trivalent”, and “multivalent” denote the presence of two binding domains, three binding domains, and multiple binding domains, respectively. The multispecific, e.g. bispecific, antibodies of the invention may have more than one binding domain capable of binding to each target antigen (i.e., the antibody is trivalent or multivalent). In preferred embodiments, the multispecific, e.g. bispecific, antibodies of the invention have more than one binding domain capable of binding to the same epitope of each target antigen. In some embodiments, the multispecific, e.g. bispecific, antibodies of the invention have more than one binding domain capable of binding to different epitopes on each target antigen.

The multispecific, e.g. bispecific, antibodies of the invention may be bivalent, trivalent or tetravalent. In preferred embodiments, the multispecific, e.g. bispecific, antibody is trivalent, preferably wherein the trivalent antibody is bivalent for BCMA. Thus, the bispecific antibody may be trivalent, wherein the trivalent antibody is bivalent for BCMA.

The multispecific, e.g. bispecific, antibodies can be full length from a single species, or can be chimerized or humanized. For an antibody with more than two antigen-binding domains, some binding domains may be identical, as long as the protein has binding domains for two different antigens.

The multispecific, e.g. bispecific, antibodies of the invention can have a bispecific heterodimeric format. In some embodiments, the bispecific antibody comprises two different heavy chains and two different light chains. In other embodiments, the multispecific, e.g. bispecific, antibody comprises two identical light chains and two different heavy chains. In some embodiments, in the multispecific, e.g. bispecific, antibodies of the invention one of the two pairs of heavy chain and light chain (HC/LC) specifically binds to CD3 and the other one specifically binds to BCMA.

In embodiments in which the bispecific antibodies of the invention are bivalent, they may comprise one anti -BCMA antibody and one anti-CD3 antibody (referred to herein as the “1+1” format).

In embodiments in which the BCMA and CD3 antibodies are Fabs, the bivalent bispecific antibodies in the 1+1 format may have the format: CD3 Fab - BCMA Fab (i.e. when no Fc is present). Alternatively, the bispecific antibodies may have the format: Fc - CD3 Fab - BCMA Fab; Fc- BCMA Fab - CD3 Fab; or BCMA Fab - Fc - CD3 Fab (i.e. when an Fc is present). In preferred embodiments, the bivalent bispecific antibodies have the format BCMA Fab - Fc - CD3 Fab.

“CD3 Fab - BCMA Fab” means that the CD3 Fab is bound via its N-terminus to the C-terminus of the BCMA Fab.

“Fc - BCMA Fab - CD3 Fab” means that the BCMA Fab is bound via its C-terminus to the N- terminus of the Fc, and the CD3 Fab is bound via its C-terminus to the N-terminus of the BCMA Fab.

“Fc - CD3 Fab - BCMA Fab” means that the CD3 Fab is bound via its C-terminus to the N-terminus of the Fc, and the BCMA Fab is bound via its C-terminus to the N-terminus of the CD3 Fab. “BCMA Fab - Fc - CD3 Fab” means that the BCMA and CD3 Fab fragments are bound via their C -terminus to the N-terminus of the Fc.

In embodiments in which the bispecific antibodies of the invention are trivalent, they may comprise two anti-BCMA antibodies and one anti-CD3 antibody (referred to herein as the “2+1” format).

In embodiments in which the BCMA and CD3 antibodies are Fabs, the trivalent bispecific antibodies in the 2+1 format may have the format: CD3 Fab - BCMA Fab - BCMA Fab; or BCMA Fab - CD3 Fab - BCMA Fab (i.e. when no Fc is present). Alternatively, the bispecific antibodies may have the format: BCMA Fab - Fc - CD3 Fab - BCMA Fab; BCMA Fab - Fc - BCMA Fab - CD3 Fab; or CD3 Fab - Fc - BCMA Fab - BCMA Fab (i.e. when an Fc is present). In preferred embodiments, the trivalent bispecific antibodies have the format BCMA Fab - Fc - CD3 Fab - BCMA Fab.

“CD3 Fab - BCMA Fab - BCMA Fab” means that the CD3 Fab is bound via its C-terminus to the N-terminus of the first BCMA Fab, and the first BCMA Fab is bound via its C-terminus to the N- terminus of the second BCMA Fab.

“BCMA Fab - CD3 Fab - BCMA Fab” means that the first BCMA Fab is bound via its C-terminus to the N-terminus of the CD3 Fab, and the CD3 Fab is bound via its C-terminus to the N-terminus of the second BCMA Fab.

“BCMA Fab - Fc - CD3 Fab - BCMA Fab” means that the first BCMA Fab and the CD3 Fab are bound via their C-terminus to the N-terminus of the Fc, and the second BCMA Fab is bound via its C-terminus to the N-terminus of the CD3 Fab.

“BCMA Fab - Fc - BCMA Fab - CD3 Fab” means that the first BCMA Fab and the second BCMA Fab are bound via their C-terminus to the N-terminus of the Fc, and the CD3 Fab is bound via its C-terminus to the N-terminus of the second BCMA Fab.

“CD3 Fab - Fc - BCMA Fab - BCMA Fab” means that the CD3 Fab and the first BCMA Fab are bound via their C-terminus to the N-terminus of the Fc, and the second BCMA Fab is bound via its C-terminus to the N-terminus of the first BCMA Fab.

In some embodiments, the bispecific antibodies of the invention may comprise not more than one BCMA Fab specifically binding to BCMA, and not more than one CD3 Fab specifically binding to CD3 and not more than one Fc part. In some embodiments, the bispecific antibody comprises not more than one CD3 Fab specifically binding to CD3, not more than two BCMA Fabs specifically binding to BCMA and not more than one Fc part. In some embodiments, not more than one CD3 Fab and not more than one BCMA Fab are linked to the Fc part and linking is performed via C-terminal binding of the Fab(s) to the hinge region of the Fc part. In some embodiments, the second BCMA Fab is linked via its C-terminus either to the N-terminus of the CD3 Fab or to the hinge region of the Fc part and is therefore between the Fc part of the bispecific antibody and the CD3 Fab.

In embodiments comprising two BCMA Fabs, the BCMA Fabs are preferably derived from the same antibody and are preferably identical in the CDR sequences, variable domain sequences VH and VL and/or the constant domain sequences CHI and CL. Preferably, the amino acid sequences of the two BCMA Fab are identical.

The bispecific antibodies of the invention can also comprise scFvs instead of the Fabs. Thus, in some embodiments, the bispecific antibodies have any one of the above formats, wherein each Fab is replaced with a corresponding scFv.

The components, e.g. the Fab fragments, of the bispecific antibodies of the invention may be chemically linked together by the use of an appropriate linker according to the state of the art. In preferred embodiments, a (Gly4-Serl) 2 linker is used (Desplancq DK et al., Protein Eng. 1994 Aug; 7(8): 1027-33 andMackM. et ah, PNAS July 18, 1995 vol. 92 no. 15 7021-7025). “Chemically linked” (or “linked”) as used herein means that the components are linked by covalent binding. As the linker is a peptidic linker, such covalent binding is usually performed by biochemical recombinant means. For example, the binding may be performed using a nucleic acid encoding the VL and/or VH domains of the respective Fab fragments, the linker and the Fc part chain if the antibody comprises an Fc.

In the event that a linker is used, this linker may be of a length and sequence sufficient to ensure that each of the first and second domains can, independently from each other, retain their differential binding specificities.

Antibody sequences

In some embodiments, the multispecific (e.g. bispecific) antibody comprises an anti-BCMA antibody, or antigen binding fragment thereof, comprising a CDR3H region of SEQ ID NO: 17 and a CDR3L region of SEQ ID NO:20 and a CDR1H, CDR2H, CDR1L, and CDR2L region combination selected from the group of a) CDR1H region of SEQ ID NO:21 and CDR2H region of SEQ ID NO:22, CDR1L region of SEQ ID NO: 23, and CDR2L region of SEQ ID NO: 24, b) CDR1H region ofSEQ ID NO:21 and CDR2H region of SEQ ID NO:22, CDR1L region of SEQ ID NO:25, and CDR2L region of SEQ ID NO:26, c) CDR1H region of SEQ ID NO:21 and CDR2H region of SEQ ID NO:22, CDR1L region of SEQ ID NO:27, and CDR2L region of SEQ ID NO:28, d) CDR1H region of SEQ ID NO: 29 and CDR2H region of SEQ ID NO: 30, CDR1L region of SEQ ID NO: 31, and CDR2L region of SEQ ID NO: 32, e) CDR1H region of SEQ ID NO:34 and CDR2H region of SEQ ID NO:35, CDR1L region of SEQ ID NO: 31, and CDR2L region of SEQ ID NO: 32, f) CDR1H region of SEQ ID NO:36 and CDR2H region of SEQ ID NO:37, CDR1L region of SEQ ID NO: 31, and CDR2L region of SEQ ID NO: 32, and g) CDR1H region of SEQ ID NO: 15 and CDR2H region of SEQ ID NO: 16, CDR1L region of SEQ ID NO: 18, and CDR2L region of SEQ ID NO: 19.

In preferred embodiments, the multispecific (e.g. bispecific) antibody comprises an anti-BCMA antibody, or antigen binding fragment thereof, comprising a VH region comprising a CDR1H region of SEQ ID NO:21, a CDR2H region of SEQ ID NO:22 and a CDR3H region of SEQ ID NO: 17 and a VL region comprising a CDR3L region of SEQ ID NO:20 and a CDR1L and CDR2L region combination selected from the group of: l) CDR1L region of SEQ ID NO:27 and CDR2L region of SEQ ID NO:28; h) CDR1L region of SEQ ID NO:23 and CDR2L region of SEQ ID NO:24; or hi) CDR1L region of SEQ ID NO:25 and CDR2L region of SEQ ID NO:26.

In particularly preferred embodiments, the multispecific (e.g. bispecific) antibody comprises an anti-BCMA antibody, or antigen binding fragment thereof, comprising a VH region comprising a CDR1H region of SEQ ID NO:21, a CDR2H region of SEQ ID NO:22 and a CDR3H region of SEQ ID NO: 17 and a VL region comprising a CDR1L region of SEQ ID NO:27, a CDR2L region of SEQ ID NO:28 and a CDR3L region of SEQ ID NO:20.

In some embodiments, the multispecific (e.g. bispecific) antibody comprises an anti-BCMA antibody, or antigen binding fragment thereof, comprising a VH and a VL selected from the group consisting of: a) a VH region of SEQ ID NO: 10 and a VL region of SEQ ID NO: 12, b) a VH region of SEQ ID NO: 10 and a VL region of SEQ ID NO: 13, c) a VH region of SEQ ID NO: 10 and a VL region of SEQ ID NO: 14, d) a VH region of SEQ ID NO:38 and a VL region of SEQ ID NO: 12, e) a VH region of SEQ ID NO:39 and a VL region of SEQ ID NO: 12, f) a VH region of SEQ ID NO:40 and a VL region of SEQ ID NO: 12, or g) a VH region of SEQ ID NO: 9 and a VL region of SEQ ID NO: 11.

In particularly preferred embodiments, the anti-BCMA antibody, or antigen binding fragment thereof, comprises a VH region of SEQ ID NO: 10 and a VL region of SEQ ID NO: 14.

In some embodiments, the multispecific (e.g. bispecific) antibody comprises an anti-CD3 antibody, or antigen binding fragment thereof.

Examples of anti-CD3 antibodies include OKT3, TR66, APA 1/1, SP34, CH2527, WT31, 7D6, UCHT-1, Leu-4, BC-3, H2C, HuM291 (visilizumab), Hu291 (PDL), ChAglyCD3 (Otehxizumab), hOKT3yl (Ala-Ala) (Teplizumab) and NI-0401 (Foralumab).

The first anti-CD3 antibody generated was OKT3 (muromonab-CD3), a murine antibody binding to the CD3s domain. Subsequent anti-CD3 antibodies include humanized or human antibodies, and engineered antibodies, for example antibodies comprising modified Fc regions.

Anti-CD3 antibodies may recognise an epitope on a single polypeptide chain, for example APA 1/1 or SP34 (Yang SJ, The Journal of Immunology (1986) 137; 1097-1100), or a conformational epitope located on two or more subunits of CD3, for example WT31, 7D6, UCHT-1 (see W02000041474) and Leu-4. Clinical trials have been carried out using several anti-CD3 antibodies, including BC-3 (Anasetti et al, Transplantation 54: 844 (1992) and H2C (WO2008119567A2). Anti-CD3 antibodies in clinical development include HuM291 (visilizumab) (Norman et al, Transplantation. 2000 Dec 27;70(12): 1707-12.) Hu291 (PDL), ChAglyCD3 (Otehxizumab) (H Waldmann), hOKT3yl (Ala-Ala) (Teplizumab) (J Bluestone and Johnson and Johnson) and (NI- 0401) Foralumab.

Any anti-CD3 antibody or antigen-binding fragment thereof may be suitable for use in the multispecific (e.g. bispecific) antibodies of the present invention. For example, the multispecific (e.g. bispecific) antibodies may comprise an anti-CD3 antibody selected from OKT3, TR66, APA 1/1, SP34, CH2527, WT31, 7D6, UCHT-1, Leu-4, BC-3, H2C, HuM291 (visilizumab), Hu291 (PDL), ChAglyCD3 (Otelixizumab), hOKT3yl (Ala-Ala) (Teplizumab) andNI-0401 (Foralumab). In some embodiments, the multispecific (e.g. bispecific) antibody of the invention comprises a humanized SP34 antibody or antigen-binding fragment thereof.

In some preferred embodiments, the anti-CD3 antibody, or antigen binding fragment thereof, may be derived from SP34 and may have similar sequences and the same properties with regard to epitope binding as antibody SP34.

In some embodiments, the multispecific (e.g. bispecific) antibody comprises an anti-CD3 antibody, or antigen binding fragment thereof, comprising a variable domain VH comprising the heavy chain CDRs of SEQ ID NO: 1, 2 and 3 as respectively heavy chain CDR1H, CDR2H and CDR3H and a variable domain VL comprising the light chain CDRs of SEQ ID NO: 4, 5 and 6 as respectively light chain CDR1L, CDR2L and CDR3L. In some embodiments, the multispecific (e.g. bispecific) antibody comprises an anti-CD3 antibody, or antigen binding fragment thereof, comprising the variable domains of SEQ ID NO:7 (VH) and SEQ ID NO:8 (VL).

In some embodiments, the multispecific (e.g. bispecific) antibody comprises an anti-BCMA antibody, or antigen binding fragment thereof, comprising a CDR3H region of SEQ ID NO: 17 and a CDR3L region of SEQ ID NO:20 and a CDR1H, CDR2H, CDR1L, and CDR2L region combination selected from the group of: a) CDR1H region of SEQ ID NO:21 and CDR2H region of SEQ ID NO:22, CDR1L region of SEQ ID NO:23, and CDR2L region of SEQ ID NO:24, b) CDR1H region ofSEQ ID NO:21 and CDR2H region of SEQ ID NO:22, CDR1L region of SEQ ID NO:25, and CDR2L region of SEQ ID NO:26, c) CDR1H region of SEQ ID NO:21 and CDR2H region of SEQ ID NO:22, CDR1L region of SEQ ID NO: 27, and CDR2L region of SEQ ID NO: 28, d) CDR1H region of SEQ ID NO: 29 and CDR2H region of SEQ ID NO: 30, CDR1L region of SEQ ID NO: 31, and CDR2L region of SEQ ID NO: 32, e) CDR1H region of SEQ ID NO:34 and CDR2H region of SEQ ID NO:35, CDR1L region of SEQ ID NO:31, and CDR2L region of SEQ ID NO:32, f) CDR1H region of SEQ ID NO:36 and CDR2H region of SEQ ID NO:37, CDR1L region of SEQ ID NO: 31, and CDR2L region of SEQ ID NO: 32, and g) CDR1H region of SEQ ID NO: 15 and CDR2H region of SEQ ID NO: 16, CDR1L region of SEQ ID NO: 18, and CDR2L region of SEQ ID NO: 19, and an anti-CD3 antibody, or antigen binding fragment thereof, comprising a CDR1H region of SEQ ID NO:l, a CDR2H region of SEQ ID NO:2, a CDR3H region of SEQ ID NO:3, a CDR1L region of SEQ ID NO:4, a CDR2L region of SEQ ID NO: 5 and a CDR3L region of SEQ ID NO:6.

In particularly preferred embodiments, the multispecific (e.g. bispecific) antibody comprises: an anti-BCMA antibody, or antigen binding fragment thereof, comprising a VH region comprising a CDR1H region of SEQ ID NO:21, a CDR2H region of SEQ ID NO:22 and a CDR3H region of SEQ ID NO: 17 and a VL region comprising a CDR1L region of SEQ ID NO:27, a CDR2L region of SEQ ID NO:28 and a CDR3L region of SEQ ID NO:20; and an anti-CD3 antibody, or antigen binding fragment thereof, comprising a CDR1H region of SEQ ID NO:l, a CDR2H region of SEQ ID NO:2, a CDR3H region of SEQ ID NO:3, a CDR1L region of SEQ ID NO:4, a CDR2L region of SEQ ID NO:5 and a CDR3L region of SEQ ID NO:6.

In some embodiments, the multispecific (e.g. bispecific) antibody comprises an anti-BCMA antibody, or antigen binding fragment thereof, comprising a VH and a VL selected from the group consisting of: a) a VH region of SEQ ID NO: 10 and a VL region of SEQ ID NO: 12, b) a VH region of SEQ ID NO: 10 and a VL region of SEQ ID NO: 13, c) a VH region of SEQ ID NO: 10 and a VL region of SEQ ID NO: 14, d) a VH region of SEQ ID NO:38 and a VL region of SEQ ID NO: 12, e) a VH region of SEQ ID NO:39 and a VL region of SEQ ID NO: 12, f) a VH region of SEQ ID NO: 40 and a VL region of SEQ ID NO: 12, and an anti-CD3 antibody, or antigen binding fragment thereof, comprising a VH region of SEQ ID NO:7 and a VL region of SEQ ID NO: 8.

In particularly preferred embodiments, the multispecific (e.g. bispecific) antibody comprises an anti-BCMA antibody, or antigen binding fragment thereof, comprising a VH region of SEQ ID NO: 10 and a VL region of SEQ ID NO: 14, and an anti-CD3 antibody, or antigen binding fragment thereof, comprising a VH region of SEQ ID NO:7 and a VL region of SEQ ID NO: 8. Fc

The multispecific, e.g. bispecific, antibodies of the invention may have an Fc or may not have an Fc. In preferred embodiments, the multispecific antibodies of the invention comprise an Fc, preferably a human Fc.

In certain embodiments, the Fc is a variant Fc, e.g., an Fc sequence that has been modified (for example by amino acid substitution, deletion and/or insertion) relative to a parent Fc sequence (for example an unmodified Fc polypeptide that is subsequently modified to generate a variant), to provide desirable structural features and/or biological activity,

Accordingly, the multispecific antibodies, e.g. bispecific antibodies, of the invention may comprise an Fc comprising one or more modifications, typically to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and/or antigen- dependent cellular cytotoxicity. The Fc may be linked to the anti-BCMA and/or anti-CD3 Fab fragments in the antibodies of the invention.

The presence of an Fc has the advantage of extending the elimination half-life of the antibody. The antibodies, e.g. bispecific antibodies, of the invention may have an elimination half-life in mice or cynomolgus monkeys, preferably cynomolgus monkeys, of longer than 12 hours, preferably 3 days or longer. In some embodiments, the antibodies, e.g. bispecific antibodies, of the invention have an elimination half-life of about 1 to 12 days, which allows at least once or twice/week administration.

Reduced effector function

Preferably, the bispecific antibodies of the invention comprise an Fc region (e.g. of IgGl subclass) that comprises modifications to avoid FcR and Clq binding and minimize ADCC/CDC. This provides the advantage that the bispecific antibody mediates its tumor cell killing efficacy purely by the powerful mechanism of effector cell, e.g. T cell, redirection/activation. Therefore, additional mechanisms of action, such as effects on the complement system and on effector cells expressing FcR, are avoided and the risk of side-effects, such as infusion-related reactions, is decreased.

In preferred embodiments, the antibodies, e.g. bispecific antibodies, of the invention comprise an IgG, particularly IgGl, Fc region comprising the modifications L234A, L235A and P329G (numbered according to EU numbering).

Heterodimerization The multispecific, e.g. bispecific, antibodies of the invention may be heteromultimeric antibodies. Such heteromultimeric antibodies may comprise modifications in regions involved in interactions between antibody chains to promote correct assembly of the antibodies.

For example, the bispecific antibodies of the invention may comprise an Fc having one or more modification(s) in the CH2 and CH3 domain to enforce Fc heterodimerization. Alternatively or in addition, the bispecific antibodies of the invention may comprise modifications in the CHI and CL region to promote preferential pairing between the heavy chain and light chain of a Fab fragment.

A number of strategies exist for promoting heterodimerization. These strategies may include the introduction of asymmetric complementary modifications into each of two antibody chains, such that both chains are compatible with each other and thus able to form a heterodimer, but each chain is not able to dimerize with itself. Such modifications may encompass insertions, deletions, conservative and non-conservative substitutions and rearrangements.

Heterodimerization may be promoted by the introduction of charged residues to create favourable electrostatic interactions between a first antibody chain and a second antibody chain. For example, one or more positively charged amino acids amino acid may be introduced into a first antibody chain, and one or more negatively charged amino acids may be introduced into a corresponding positions in a second antibody chain

Alternatively or in addition, heterodimerization may be promoted by the introduction of steric hindrance between contacting residues. For example, one or more residues with a bulky side chain may be introduced into a first antibody chain, and a one or more residues able to accommodate the bulky side chain may be introduced into the second antibody chain.

Alternatively or in addition, heterodimerization may be promoted by the introduction of one or more modification(s) to the hydrophilic and hydrophobic residues at the interface between chains, in order make heterodimer formation more entropically and enthalpically favourable than homodimer formation.

A further strategy for promoting heterodimerization is to rearrange portions of the antibody chains such that each chain remains compatible only with a chain comprising corresponding rearrangements. For example, CrossMAb technology is based on the crossover of antibody domains in order to enable correct chain association. There are three main CrossMAb formats, these are: (i) CrossMAb Fab in which the VH and VL are exchanged and the CHI and CL are exchanged; (ii) CrossMAb vll VL in which the VH and VL are exchanged; and (iii) CrossMAb CH1 CL in which the CHI and CL are exchanged (Klein et al., 2016. MABS, 8(6): 1010-1020).

In some embodiments, the bispecific antibodies of the invention may comprise an exchange of the VH and VL. In some embodiments, the antibodies, e.g. bispecific antibodies, of the invention may comprise an exchange of the CHI and CL. In some embodiments, the antibodies, e.g. bispecific antibodies, of the invention may comprise an exchange of the VH and VL and an exchange of the CHI and CL.

In preferred embodiments, the antibodies, e.g. bispecific antibodies, of the invention comprise an exchange of the VH and VL.

Other approaches to promoting heterodimerization include the use of a strand exchange engineered domain (SEED) (Davis et al., 2010. Protein Eng Des Sel, 23 (4); 195- 202).

A combination of the above strategies may be used to maximise the efficiency of assembly while minimising the impact on antibody stability.

Fc heterodimerization

In some embodiments, multispecific antibodies, e.g. bispecific antibodies, of the invention may have a heterodimeric Fc, for example they may comprise one heavy chain originating from an anti- BCMA antibody, and one heavy chain originating from an anti-CD3 antibody.

The antibodies, e.g. bispecific antibodies, of the invention may comprise a heterodimeric Fc which comprises one or more modification(s) which promotes the association of the first CH2 and/or CH3 domain with the second CH2 and/or CH3 domain. In preferred embodiments, the one or more modification(s) promote the association of the first CH3 domain with the second CH3 domain, for example by resulting in asymmetric modifications to the CH3 domain. The one or more modification(s) may comprise modifications selected from amino acid insertions, deletions, conservative and non-conservative substitutions and rearrangements, and combinations thereof.

Typically the first CH3 domain and the second CH3 domain are both engineered in a complementary manner so that each CH3 domain (or the heavy chain comprising it) can no longer homodimerize with itself but is forced to heterodimerize with the complementary engineered other CH3 domain (so that the first and second CH3 domain heterodimerize and no homodimers between the two first or the two second CH3 domains are formed). The multispecific, e.g. bispecific, antibodies of the invention may comprise an Fc having one or more of “knob-into-holes” modification(s), which are described in detail with several examples in e.g. WO 96/027011, Ridgway, J.B., et al., Protein Eng. 9 (1996) 617-621, Merchant, A.M. et al., Nat. Biotechnol. 16 (1998) 677-68, and WO 98/050431.

In this method, the interaction surfaces of the two CH3 domains are altered to increase the heterodimerization of both Fc chains containing these two CH3 domains. One of the two CH3 domains (of the two Fc chains) can be the "knob", while the other is the "hole".

Accordingly, the bispecific antibodies of the invention may comprise two CH3 domains, wherein the first CH3 domain of the first Fc chain and the second CH3 domain of the second Fc chain each meet at an interface which comprises an original interface between the antibody CH3 domains, wherein said interface is altered to promote the formation of the antibody.

In some embodiments:

(i) the CH3 domain of one Fc chain is altered, so that within the original interface of the CH3 domain of the one Fc chain that meets the original interface of the CH3 domain of the other Fc chain, an amino acid residue is replaced with an amino acid residue having a larger side chain volume, thereby generating a protuberance within the interface of the CH3 domain of one Fc chain which is positionable in a cavity within the interface of the CH3 domain of the other Fc chain; and ii) the CH3 domain of the other Fc chain is altered, so that within the original interface of the CH3 domain of the other Fc chain that meets the original interface of the CH3 domain of the one Fc chain, an amino acid residue is replaced with an amino acid residue having a smaller side chain volume, thereby generating a cavity within the interface of the CH3 domain of the other Fc chain within which a protuberance within the interface of the CH3 domain of the one Fc chain is positionable.

Preferably, said amino acid residue having a larger side chain volume is selected from the group consisting of arginine (R), phenylalanine (F), tyrosine (Y), tryptophan (W).

In some embodiments, the multispecific, e.g. bispecific, antibodies of the invention comprise a first CH3 domain comprising modification(s) at positions T366, L368 and Y407, e.g. T366S, L368A, and Y407V (numbered according to EU numbering).

In some embodiments, the multispecific, e.g. bispecific, antibodies of the invention comprise a second CH3 domain comprising a modification at position T366 (“knob modification”), e.g. T366W (numbered according to EU numbering).

In particularly preferred embodiments, the multispecific, e.g. bispecific, antibodies of the invention comprise a first CH3 domain comprising the modifications T366S, L368A, and Y407V, or conservative substitutions thereof, and a second CH3 domain comprising the modification T366W, or a conservative substitution thereof (numbered according to EU numbering).

In one embodiment, the multispecific, e.g. bispecific, antibodies of the invention comprise a first CH3 domain comprising the modification set forth in Table 2 and a second CH3 domain comprising the modifications set forth in Table 2. Table 2: “Knob-in to-holes” modification

Other techniques for CH3 modifications to enforce heterodimerization are contemplated as alternatives of the invention and are described e.g. in WO96/27011, W098/050431, EP1870459, W02007/110205, W02007/147901, W02009/089004, W02010/129304, WO2011/90754, WO2011/143545, WO2012/058768, WO2013/157954, WO2013/157953, and WO2013/096291.

In some embodiments, the bispecific antibody according to the invention is of IgG2 isotype and the heterodimerization approach described in W02010/129304 can be used.

Other Fc modifications

In some embodiments, the bispecific antibodies of the invention may comprise an Fc, wherein both CH3 domains are altered by the introduction of cysteine (C) as the amino acid in the corresponding positions of each CH3 domain such that a disulphide bridge between both CH3 domains can be formed. The cysteines may be introduced at position 349 in one of the CH3 domains and at position 354 in the other CH3 domain (numbered according to EU numbering).

Preferably, the cysteine introduced at position 354 is in the first CH3 domain and the cysteine introduced at position 349 is in the second CH3 domain (numbered according to EU numbering).

The Fc may comprise modifications, such as D356E, L358M, N384S, K392N, V397M, and V422I (numbered according to EU numbering). Preferably, both CH3 domains comprise D356E and L358M (numbered according to EU numbering).

Light and heavy chain heterodimerization

In the multispecific, e.g. bispecific, antibodies of the invention, one or more of the immunoglobulin heavy chains and light chains may comprise one or more modification(s), e.g. amino acid modifications that are capable of promoting preferential pairing of a specific heavy chain with a specific light chain when heavy chains and light chains are co-expressed or co-produced. Such modifications can provide considerably improved production/purification without changing biological properties such as binding to BCMA. In particular, by introduction of one or more modification(s) such as amino acid exchanges, light chain mispairing and the formation of side products in production can be significantly reduced and therefore yield is increased and purification is facilitated.

The amino acid exchanges may be substitutions of charged amino acids with opposite charges (for example in the CHI/CL interface) which reduce light chain mispairing, e.g. Bence-Jones type side products.

In preferred embodiments, the one or more modification(s) assist light and heavy chain heterodimerization are amino acid modifications in the light and heavy chains outside of the CDRs.

The one or more modification(s) may be present in the anti-BCMA antibody or antigen-binding fragment thereof. Alternatively, the one or more modification(s) may be present in the anti-CD3 antibody or antigen-binding fragment thereof. In preferred embodiments, the one or more modification(s) are present in the anti-BCMA antibody or antigen-binding fragment thereof.

In some embodiments, the multispecific, e.g. bispecific, antibodies of the invention comprise an immunoglobulin heavy chain comprising a CHI domain having amino acid modifications K147E/D and K213E/D (numbered according to EU numbering) and a corresponding immunoglobulin light chain comprising a CL domain having amino acid modifications E123K/R/H and Q124K/R/H (numbered according to Kabat). Preferably, the CHI domain comprises the amino acid modifications K147E and K213E (numbered according to ELI numbering) or conservative substitutions thereof, and the corresponding CL domain comprises the amino acid modifications E123R and Q124K or conservative substitutions thereof (numbered according to Kabat). Such multispecific, e.g. bispecific, antibodies can be produced in high yield and can be easily purified.

In one embodiment, the amino acid modifications described in Table 3 can be in the BCMA antibody or in the CD3 antibody.

In one embodiment, the bispecific antibodies of the invention are bivalent, and comprise one anti- BCMA antibody or antigen-binding fragment thereof and one anti-CD3 antibody or antigen-binding fragment thereof (the “1+1” format), wherein:

(a) the BCMA antibody or antigen-binding fragment thereof (e.g. BCMA Fab) comprises a CHI domain having amino acid modifications set forth in Table 3 and a corresponding CL domain having the amino acid modifications Table 3; or

(b) the CD3 antibody or antigen-binding fragment thereof (e.g. CD3 Fab) comprises a CHI domain having amino acid modifications set forth in Table 3 and a corresponding CL domain having the amino acid modifications Table 3.

In one embodiment, the bispecific antibodies of the invention are trivalent and comprise two anti- BCMA antibodies or antigen-binding fragments thereof and one anti-CD3 antibody or antigen binding fragment thereof (the “2+1” format), wherein:

(a) one or both BCMA antibodies or antigen-binding fragments thereof (e.g. BCMA Fabs) comprises a CHI domain having amino acid modifications set forth in Table 3 and a corresponding CL domain having the amino acid modifications Table 3; or

(b) the CD3 antibody (e.g. CD3 Fab) comprises a CHI domain having amino acid modifications set forth in Table 3 and a corresponding CL domain having the amino acid modifications Table 3.

In particular, each BCMA antibody (e.g. BCMA Fab) may comprise a CHI domain having amino acid modifications set forth in Table 3 and a corresponding CL domain having the amino acid modifications Table 3. Table 3: Light and heavy chain heterodimerization modifications

In a preferred embodiment, the multispecific, e.g. bispecific, antibodies of the invention comprise the modifications set forth in Table 3 in combination with the modifications set forth in Table 2. Thus, in one embodiment, the bispecific antibodies of the invention are bivalent, and comprise:

(a) one anti-BCMA antibody or antigen-binding fragment thereof and one anti-CD3 antibody or antigen-binding fragment thereof (the “1+1” format), wherein (i) the BCMA antibody or antigen-binding fragment thereof (e.g. BCMA Fab) comprises a CHI domain that comprises the amino acid modifications K147E and K213E, and a corresponding CL domain that comprises the amino acid modifications E123R and Q124K (i.e. the modifications set forth in Table 3), or (ii) the CD3 antibody or antigen-binding fragment thereof (e.g. CD3 Fab) comprises a CHI domain that comprises the amino acid modifications K147E and K213E, and a corresponding CL domain that comprises the amino acid modifications E123R and Q124K (i.e. the modifications set forth in Table 3); and

(b) a first CH3 domain comprising the modifications T366S, L368A, and Y407V, and a second CH3 domain comprising the modification T366W (i.e. the modifications set forth in Table 2)·

In one embodiment, the bispecific antibodies of the invention are trivalent and comprise:

(a) two anti-BCMA antibodies or antigen-binding fragments thereof and one anti-CD3 antibody or antigen-binding fragment thereof (the “2+1” format), wherein (i) one or both

BCMA antibodies or antigen-binding fragments thereof (e.g. BCMA Fabs) comprises a CHI domain that comprises the amino acid modifications K147E and K213E, and a corresponding CL domain that comprises the amino acid modifications E123R and Q124K (i.e. the modifications set forth in Table 3), or (ii) the CD3 antibody or antigen-binding fragment thereof ( e.g . CD3 Fab) comprises a CHI domain that comprises the amino acid modifications K147E and K213E, and a corresponding CL domain that comprises the amino acid modifications E123R and Q124K (i.e. the modifications set forth in Table 3); and (b) a first CH3 domain comprising the modifications T366S, L368A, and Y407V, and a second CH3 domain comprising the modification T366W (i.e. the modifications set forth in Table 2)·

In particular, each BCMA antibody (e.g. BCMA Fab) may comprise a CHI domain having amino acid modifications set forth in Table 3 and a corresponding CL domain having the amino acid modifications Table 3. In preferred embodiments, the first Fc chain is bound at the N-terminus of the Fc to the C-terminus of the first anti-BCMA antibody, and the second Fc chain is bound at the N-terminus of the Fc to the C-terminus of the anti-CD3 antibody.

The multispecific, e.g. bispecific, antibodies of the invention may additionally comprise an amino acid substitution at position 49 of the VL region selected from the group of amino acids tyrosine (Y), glutamic acid (E), serine (S), and histidine (H) and/or an amino acid substitution at position 74 of the VL region that is threonine (T) or alanine (A).

CrossMAb

The multispecific, e.g. bispecific, antibodies of the invention may comprise CrossMAb technology. CrossMAb technology is based on the crossover of antibody domains in order to enable correct chain association. It is used to facilitate multispecific antibody formation. There are three main CrossMAb formats, these are: (i) CrossMAb Fab in which the VH and VL are exchanged and the CHI and CL are exchanged; (ii) CrossMAb 1 ^ 11 ^ in which the VH and VL are exchanged; and (iii) CrossMAb CH1 CL in which the CHI and CL are exchanged (Klein et al., 2016. MABS, 8(6): 1010- 1020).

CrossMAb technology is known in the state of the art. Bispecific antibodies wherein the variable domains VL and VH or the constant domains CL and CHI are replaced by each other are described in W02009080251 and W02009080252.

In one or more of the antibodies or antigen-binding fragments within the multispecific, e.g. bispecific, antibodies of the invention, the variable domains VL and VH or the constant domains CL and CHI may be replaced by each other. In some embodiments, the antibodies, e.g. bispecific antibodies, of the invention may comprise an exchange of the VH and VL and an exchange of the CHI and CL. Thus, the multispecific, e.g. bispecific, antibodies of the invention may comprise a crossover light chain and a crossover heavy chain. As used herein, a "crossover light chain" is a light chain that may comprise a VH-CL, a VL-CH1 or a VH-CH1. A "crossover heavy chain" as used herein is a heavy chain that may comprise a VL-CH1 , a VH-CL or a VL-CL.

In some aspects, there is provided a multispecific, e.g. bispecific, antibody comprising an anti- BCMA antibody of the invention, or an antigen-binding fragment thereof, and an anti-CD3 antibody, or antigen-binding fragment thereof, wherein the multispecific, e.g. bispecific, antibody comprises:

(a) a light chain and a heavy chain of an antibody specifically binding to CD3; and

(b) a light chain and heavy chain of an antibody specifically binding to BCMA, wherein the variable domains VL and VH and/or the constant domains CL and CHI are replaced by each other in (i) the anti-BCMA antibody; and/or (ii) the anti-CD3 antibody.

In some embodiments, the variable domains VL and VH or the constant domains CL and CHI of the anti-CD3 antibody or antigen binding fragment thereof are replaced by each other. More preferably, the variable domains VL and VH of the anti-CD3 antibody or antigen binding fragment thereof are replaced by each other.

In embodiments in which the bispecific antibodies in the 1+1 format have the format: CD3 Fab - BCMA Fab (i.e. when no Fc is present); Fc - CD3 Fab - BCMA Fab; Fc- BCMA Fab - CD3 Fab; or BCMA Fab - Fc - CD3 Fab, the bispecific antibodies may comprise the CrossMAb format, e.g. CrossMAb Fab , CrossMAb VH VL or CrossMAb CH1 CL . The BCMA Fab may have the CrossMAb format, e.g. CrossMAb Fab , CrossMAb vl l VL or CrossMAb CH1 CL . Alternatively, the CD3 Fab may have the CrossMAb format, e.g. CrossMAb Fab , CrossMAb vl l VL or CrossMAb CH1 CL . In preferred embodiments, the CD3 Fab of the bispecific antibody comprises the CrossMAb vl l VL format.

It is especially preferred for the bispecific antibodies of the invention having the 2+1 format to comprise CrossMAb technology. Thus, in embodiments in which the trivalent bispecific antibodies in the 2+1 format have the format: CD3 Fab - BCMA Fab - BCMA Fab; BCMA Fab - CD3 Fab - BCMA Fab (i.e. when no Fc is present); BCMA Fab - Fc - CD3 Fab - BCMA Fab; BCMA Fab - Fc - BCMA Fab - CD3 Fab; or CD3 Fab - Fc - BCMA Fab - BCMA Fab, the bispecific antibodies may comprise the CrossMAb format, e.g. CrossMAb Fab , CrossMAb VH VL or CrossMAb CH1 CL . The BCMA Fab may have the CrossMAb format, e.g. CrossMAb Fab , CrossMAb vl l VL or CrossMAb CH1 CL . Alternatively, the CD3 Fab may have the CrossMAb format, e.g. CrossMAb Fab , CrossMAb 1 ^ 1 ^ or CrossMAb CH1 CL . In preferred embodiments, the CD3 Fab of the bispecific antibody comprises the CrossMAb VH VL format.

In some embodiments, the bispecific antibodies of the invention having the 1+1 format do not comprise CrossMAb technology, i.e. neither the anti-BCMA antibody nor the anti-CD3 antibody have the variable domains VL and VH or the constant domains CL and CHI replaced by each other.

Exemplary Embodiments

Exemplary embodiments are set out in Figures 1-3.

In one embodiment, the bispecific antibodies according to the invention are bivalent bispecific antibodies comprising one Fab fragment of an anti-CD3 antibody, one Fab fragment of an anti- BCMA antibody and one Fc part according to the format BCMA Fab - Fc - CD3 Fab. The anti- BCMA Fab fragment comprises the amino acid modifications set forth in Table 3. The anti-CD3 Fab fragment comprises a light chain and heavy chain, wherein the light chain is a crossover light chain that comprises a variable domain VH and a constant domain CL, and wherein the heavy chain is a crossover heavy chain that comprises a variable domain VL and a constant domain CHI. This embodiment is illustrated in Figure 1 A.

In one embodiment, the bispecific antibodies according to the invention are bivalent bispecific antibodies comprising one Fab fragment of an anti-CD3 antibody, one Fab fragment of an anti- BCMA antibody and one Fc part according to the format BCMA Fab - Fc - CD3 Fab. The anti- CD3 Fab fragment comprises (a) a light chain and heavy chain, wherein the light chain is a crossover light chain that comprises a variable domain VH and a constant domain CL, and wherein the heavy chain is a crossover heavy chain that comprises a variable domain VL and a constant domain CHI; and also (b) the amino acid modifications set forth in Table 3. This embodiment is illustrated in Figure IB.

In one embodiment, the bispecific antibodies according to the invention are trivalent bispecific antibodies comprising one Fab fragment of an anti-CD3 antibody, two Fab fragments of an anti- BCMA antibody and one Fc part according to the format BCMA Fab - Fc - CD3 Fab - BCMA Fab. Each anti-BCMA Fab fragment comprises the amino acid modifications set forth in Table 3. The anti-CD3 Fab fragment comprises a light chain and heavy chain, wherein the light chain is a crossover light chain that comprises a variable domain VH and a constant domain CL, and wherein the heavy chain is a crossover heavy chain that comprises a variable domain VL and a constant domain CHI . This embodiment is illustrated in Figure 2A.

In one embodiment, the bispecific antibodies according to the invention are trivalent bispecific antibodies comprising one Fab fragment of an anti-CD3 antibody, two Fab fragments of an anti- BCMA antibody and one Fc part according to the format BCMA Fab - Fc - CD3 Fab - BCMA Fab. The anti-CD3 Fab fragment comprises (a) a light chain and heavy chain, wherein the light chain is a crossover light chain that comprises a variable domain VH and a constant domain CL, and wherein the heavy chain is a crossover heavy chain that comprises a variable domain VL and a constant domain CHI; and also (b) the amino acid modifications set forth in Table 3. This embodiment is illustrated in Figure 2B.

In one embodiment, the bispecific antibodies according to the invention are trivalent bispecific antibodies comprising one Fab fragment of an anti-CD3 antibody, two Fab fragments of an anti- BCMA antibody and one Fc part according to the format BCMA Fab - Fc - BCMA Fab - CD3 Fab. Each anti -BCMA Fab fragment comprises the amino acid modifications set forth in Table 3. The anti-CD3 Fab fragment comprises a light chain and heavy chain, wherein the light chain is a crossover light chain that comprises a variable domain VH and a constant domain CL, and wherein the heavy chain is a crossover heavy chain that comprises a variable domain VL and a constant domain CHI. This embodiment is illustrated in Figure 2C.

In one embodiment, the bispecific antibodies according to the invention are trivalent bispecific antibodies comprising one Fab fragment of an anti-CD3 antibody, two Fab fragments of an anti- BCMA antibody and one Fc part according to the format BCMA Fab - Fc - BCMA Fab - CD3 Fab. The anti-CD3 Fab fragment comprises (a) a light chain and heavy chain, wherein the light chain is a crossover light chain that comprises a variable domain VH and a constant domain CL, and wherein the heavy chain is a crossover heavy chain that comprises a variable domain VL and a constant domain CHI; and also (b) the amino acid modifications set forth in Table 3. This embodiment is illustrated in Figure 2D.

In one embodiment, the bispecific antibodies according to the invention are bivalent bispecific antibodies comprising one Fab fragment of an anti-CD3 antibody, one Fab fragment of an anti- BCMA antibody and one Fc part according to the format Fc - CD3 Fab - BCMA Fab. The anti- BCMA Fab fragment comprises the amino acid modifications set forth in Table 3. The anti-CD3 Fab fragment comprises a light chain and heavy chain, wherein the light chain is a crossover light chain that comprises a variable domain VH and a constant domain CL, and wherein the heavy chain is a crossover heavy chain that comprises a variable domain VL and a constant domain CHI. This embodiment is illustrated in Figure 3A.

In one embodiment, the bispecific antibodies according to the invention are bivalent bispecific antibodies comprising one Fab fragment of an anti-CD3 antibody, one Fab fragment of an anti- BCMA antibody and one Fc part according to the format Fc - CD3 Fab - BCMA Fab. The anti- CD3 Fab fragment comprises (a) a light chain and heavy chain, wherein the light chain is a crossover light chain that comprises a variable domain VH and a constant domain CL, and wherein the heavy chain is a crossover heavy chain that comprises a variable domain VL and a constant domain CHI ; and also (b) the amino acid modifications set forth in Table 3. This embodiment is illustrated in Figure 3B.

In one embodiment, the bispecific antibodies according to the invention are bivalent bispecific antibodies comprising one Fab fragment of an anti-CD3 antibody, one Fab fragment of an anti- BCMA antibody and one Fc part according to the format Fc - BCMA Fab - CD3 Fab. The anti- BCMA Fab fragment comprises the amino acid modifications set forth in Table 3. The anti-CD3 Fab fragment comprises a light chain and heavy chain, wherein the light chain is a crossover light chain that comprises a variable domain VH and a constant domain CL, and wherein the heavy chain is a crossover heavy chain that comprises a variable domain VL and a constant domain CHI. This embodiment is illustrated in Figure 3C.

In one embodiment, the bispecific antibodies according to the invention are bivalent bispecific antibodies comprising one Fab fragment of an anti-CD3 antibody, one Fab fragment of an anti- BCMA antibody and one Fc part according to the format Fc - BCMA Fab - CD3 Fab. The anti- CD3 Fab fragment comprises (a) a light chain and heavy chain, wherein the light chain is a crossover light chain that comprises a variable domain VH and a constant domain CL, and wherein the heavy chain is a crossover heavy chain that comprises a variable domain VL and a constant domain CHI; and also (b) the amino acid modifications set forth in Table 3. This embodiment is illustrated in Figure 3D.

In one embodiment, the antibodies illustrated in Figure 2 additionally comprise the modifications set forth in Table 2.

In one aspect, the bispecific antibodies according to the invention are trivalent bispecific antibodies comprising one Fab fragment of an anti-CD3 antibody, two Fab fragments of an anti-BCMA antibody and one Fc part according to the format BCMA Fab - Fc - CD3 Fab - BCMA Fab. The anti-CD3 Fab fragment comprises a light chain and heavy chain, wherein the light chain is a crossover light chain that comprises a variable domain VH and a constant domain CL, and wherein the heavy chain is a crossover heavy chain that comprises a variable domain VL and a constant domain CHI. Each anti-BCMA Fab fragment comprises a light chain and heavy chain, wherein the heavy chain comprises a CHI domain which comprises the amino acid modifications K147E and K213E (numbered according to EU numbering) and wherein the light chain comprises a corresponding CL domain which comprises the amino acid modifications E123R and Q124K (numbered according to Kabat) (i.e. the modifications set forth in Table 3). The Fc part comprises a first Fc chain and a second Fc chain, wherein the first Fc chain comprises a first constant domain CH2 and a first constant domain CH3, and the second Fc chain comprises a second constant domain CH2 and a second constant domain CH3. The first Fc chain is bound at the N-terminus of the Fc to the C-terminus of the first anti-BCMA Fab, and the second Fc chain is bound at the N-terminus of the Fc to the C-terminus of the anti-CD3 Fab. The first CH3 domain comprises the modifications T366S, L368A, and Y407V (“hole modifications”) and the second CH3 domain comprises the modification T366W (“knob modification”) (numbered according to EU numbering) (i.e. the modifications set forth in Table 2). Additionally, both Fc chains further comprise the modifications L234A, L235A and P329G, and optionally D356E and L358M (numbered according to EU numbering). Optionally, the first CH3 domain further comprises the amino acid modification S354C, and the second CH3 domain further comprises the amino acid modification Y349C (numbered according to EU numbering) such that a disulphide bridge between both CH3 domains is formed.

In some embodiments, the anti-BCMA Fab fragment comprises a CDR3H region of SEQ ID NO: 17 and a CDR3L region of SEQ ID NO:20 and a CDR1H, CDR2H, CDR1L, and CDR2L region combination selected from the group of: a) CDR1H region of SEQ ID NO:21 and CDR2H region of SEQ ID NO:22, CDR1L region of SEQ ID NO:23, and CDR2L region of SEQ ID NO:24, b) CDR1H region ofSEQ ID NO:21 and CDR2H region of SEQ ID NO:22, CDR1L region of SEQ ID NO:25, and CDR2L region of SEQ ID NO:26, c) CDR1H region of SEQ ID NO:21 and CDR2H region of SEQ ID NO:22, CDR1L region of SEQ ID NO:27, and CDR2L region of SEQ ID NO:28, d) CDR1H region of SEQ ID NO: 29 and CDR2H region of SEQ ID NO: 30, CDR1L region of SEQ ID NO: 31, and CDR2L region of SEQ ID NO: 32, e) CDR1H region of SEQ ID NO:34 and CDR2H region of SEQ ID NO:35, CDR1L region of SEQ ID NO: 31, and CDR2L region of SEQ ID NO: 32, f) CDR1H region of SEQ ID NO:36 and CDR2H region of SEQ ID NO:37, CDR1L region of SEQ ID NO: 31, and CDR2L region of SEQ ID NO: 32, and g) CDR1H region of SEQ ID NO: 15 and CDR2H region of SEQ ID NO: 16, CDR1L region of SEQ ID NO: 18, and CDR2L region of SEQ ID NO: 19, and the anti-CD3 Fab fragment comprises a CDR1H region of SEQ ID NO: 1, a CDR2H region of SEQ ID NO:2, a CDR3H region of SEQ ID NO:3, a CDR1L region of SEQ ID NO:4, a CDR2L region of SEQ ID NO:5 and a CDR3L region of SEQ ID NO:6.

In some embodiments, the anti-BCMA Fab fragment comprises a VH and a VL selected from the group consisting of: a) a VH region of SEQ ID NO: 10 and a VL region of SEQ ID NO: 12, b) a VH region of SEQ ID NO: 10 and a VL region of SEQ ID NO: 13, c) a VH region of SEQ ID NO: 10 and a VL region of SEQ ID NO: 14, d) a VH region of SEQ ID NO:38 and a VL region of SEQ ID NO: 12, e) a VH region of SEQ ID NO:39 and a VL region of SEQ ID NO: 12, f) a VH region of SEQ ID NO:40 and a VL region of SEQ ID NO: 12, or g) a VH region of SEQ ID NO: 9 and a VL region of SEQ ID NO: 11; and the anti-CD3 Fab fragment comprises a VH region of SEQ ID NO: 7 and a VL region of SEQ ID NO:8.

In further embodiments, the bispecific antibody according to the invention comprises the following SEQ ID NOs (as mentioned in Tables 4A and 5B below):

83A10-TCBcv: 45, 46, 47 (x2), 48 (Figure 2A)

21-TCBcv: 48, 49, 50, 51 (x2) (Figure 2A)

22-TCBcv: 48, 52, 53, 54 (x2) (Figure 2A)

42-TCBcv: 48, 55, 56, 57 (x2) (Figure 2A)

The term “83A10-TCBcv” as used herein refer to a bispecific antibody specifically binding to BCMA and CD3 as specified by its heavy and light chain combination of SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47 (2x), and SEQ ID NO:48, and as shown in Figure 2A and described in EP14179705.

The terms “21-TCBcv, 22-TCBcv, 42-TCBcv” as used herein refer to the respective bispecific antibodies of Mab21, as specified by its heavy and light chain combination of SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, and SEQ ID NO:51 (2x), Mab 22 as specified by its heavy and light chain combinations of SEQ ID NO:48, SEQ ID NO:52, SEQ ID NO:53, and SEQ ID NO:54 (2x), and Mab42 as specified by its heavy and light chain combination of SEQ ID NO:48, SEQ ID NO: 55, SEQ ID NO: 56, and SEQ ID NO:57-(2x), and as shown in Figure 2A and described in WO 2017/021450.

In preferred embodiments, the bispecific antibody according to the invention is 42-TCBcv. Pharmaceutical Compositions

The multispecific, e.g. bispecific, antibodies of the invention can be administered to the patient as a pharmaceutical composition. Accordingly, the present invention also provides a pharmaceutical composition comprising the multispecific, e.g. bispecific, antibodies of the invention and a pharmaceutically acceptable excipient.

The term “pharmaceutically acceptable” as used herein means approved by a regulatory agency of the Federal or a state government, or listed in the U.S. Pharmacopeia, European Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

The pharmaceutical compositions disclosed herein are for use in, but not limited to, diagnosing, detecting, or monitoring a disorder, in preventing, treating, managing, or ameliorating a disorder or one or more symptoms thereof, and/or in research. The pharmaceutical compositions disclosed herein may be suitable for veterinary uses or pharmaceutical uses in humans.

Examples of suitable excipients include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like, as well as any combination thereof. In many cases, it will be preferable to include isotonic agents, such as sugars, polyalcohols, or sodium chloride in the composition. In particular, relevant examples of suitable excipients include: (1) Dulbecco's phosphate buffered saline, pH.about.7.4, containing or not containing about 1 mg/mL to 25 mg/mL human serum albumin, (2) 0.9% saline (0.9% w/v sodium chloride (NaCl)), and (3) 5% (w/v) dextrose; and may also contain an antioxidant such as tryptamine and a stabilizing agent such as Tween 20 ® .

A person skilled in the art would understand that the appropriate choice of excipient or excipients for use with multispecific, e.g. bispecific, antibodies of the invention would depend on the desired properties of the pharmaceutical composition.

The pharmaceutical compositions or the multispecific antibodies of the invention can be administered to a patient by any appropriate systemic or local route of administration. For example, administration may be oral, buccal, sublingual, ophthalmic, intranasal, intratracheal, pulmonary, topical, transdermal, urogenital, rectal, subcutaneous, intravenous, intra-arterial, intraperitoneal, intramuscular, intracranial, intrathecal, epidural, intraventricular or intratumoral.

Pharmaceutical compositions of the invention can be formulated for administration by any appropriate means, for example by epidermal or transdermal patches, ointments, lotions, creams, or gels; by nebulizers, vaporisers, or inhalers; by injection or infusion; or in the form of capsules, tablets, liquid solutions or suspensions in water or non-aqueous media, drops, suppositories, enemas, sprays, or powders. The most suitable route for administration in any given case will depend on the physical and mental condition of the patient, the nature and severity of the disease, and the desired properties of the formulation.

Monotherapies and Combination Therapies

In some embodiments, the treatment comprises the administration of the multispecific, e.g. bispecific, antibody of the invention to the patient as a monotherapy.

In some embodiments, the treatment comprises the administration of the multispecific, e.g. bispecific, antibody of the invention to the patient as a combination therapy, wherein the combination therapy comprises the administration of the multispecific, e.g. bispecific, antibody of the invention and one or more additional therapeutic agents. The term "combination therapy" is meant to encompass administration of the selected therapeutic agents to a single patient, and is intended to include treatments in which the agents are administered by the same or different route of administration or at the same or different time.

In some embodiments, the one or more additional therapeutic agents are selected from the group consisting of thalidomide and an immunotherapeutic derivative thereof, an anti-CD38 antibody, an anti-PD-1 antibody, an anti-PD-Ll antibody, a gamma secretase inhibitor (GSI), an anti-BCMA antibody drug conjugate and anti-BCMA CAR T-cell therapy.

The term “anti-CD38 antibody” as used herein relates to an antibody specifically binding to human CD38. In an embodiment of the invention the anti-CD38 antibody is daratumumab (US20150246123). In an embodiment of the invention the anti-CD38 antibody is isatuximab (SAR650984, US8877899). In an embodiment of the invention the anti-CD38 antibody is MOR202 (WO 2012041800). In an embodiment of the invention the anti-CD38 antibody is Ab79 (US8362211). In an embodiment of the invention the anti-CD38 antibody is Abl9 (US8362211). The dosage of such anti-CD38 antibody is performed according to the state of the art and described in the respective prescribing informations. E.g. Daratumumab dosage is usually 16mg/kg (www. ema. europa. cuY

The term “thalidomide compound” or “thalidomide and an immunotherapeutic derivative” as used herein relates to 2-(2,6-dioxopiperidin-3-yl)-2,3-dihydro-lH-isoindole-l,3-dio ne and immunotherapeutic derivatives thereof. In an embodiment of the invention the thalidomide compound is selected from the group consisting of, but not limited to, thalidomide (CAS Registry Number 50-35-1), lenalidomide (CAS Registry Number 191732-72-6), pomalidomide (CAS Registry Number 19171-19-8), CC122 (CAS Registry Number 1398053-45-6) and CC-220 (CAS Registry Number 1323403-33-3) and the respective salts (preferably HC1 salts 1:1). The chemical formula of CC-122 is 2,6-piperidinedione,3-(5-amino-2-methyl-4-oxo-3(4H-quinazoli nyl), hydrochloride (1:1) and of CC-220 it is 2,6-piperidinedione, 3-[l,3-dihydro-4-[[4-(4- morpholinylmethyl)phenyl]methoxy]-l-oxo-2H-isoindol-2-yl]-, (3S)-, hydrochloride (1:1). Methods of preparing CC-220 are described, e.g., in US 20110196150, the entirety of which is incorporated herein by reference.

The dosage of thalidomide compounds is performed according to the state of the art and described in the respective prescribing informations. E.g. Revlimid® (lenalidomide) dosage is usually 25 mg once daily orally on days 1-21 of repeated 28- day cycles (www.revlimid.com) and POMALYST® (pomalidomide) dosage for the treatment of Multiple Myeloma is usually 4 mg per day taken orally on days 1-21 of repeated 28-day cycles (www.celgene.com). In one embodiment, 3-(5-amino-2- methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione is administered in an amount of about 5 to about 50 mg per day.

In one embodiment, CC-122 and CC-220 are administered in an amount of about 5 to about 25 mg per day. In another embodiment, CC-122 and CC-220 are administered in an amount of about 5, 10, 15, 25, 30 or 50 mg per day. In another embodiment, 10 or 25 mg of CC-122 and CC-220 are administered per day. In one embodiment, CC-122 and CC-220 are administered twice per day.

The term “anti -PD- 1 antibody” as used herein relates to an antibody specifically binding to human PD-1. Such antibodies are e.g. described in WO2015026634 (MK-3475, pembrolizumab), US7521051, US8008449, and US8354509. Pembrolizumab (Keytruda®, MK-3475,.) is also described in WO 2009/114335, Poole, R.M. Drugs (2014) 74: 1973; Seiwert, T.,et al., J. Clin. Oncol. 32,5s (suppl;abstr 6011). In an embodiment of the invention the PD-1 antibody is MK-3475 (WHO Drug Information, Vol. 27, No. 2, pages 161-162 (2013)) and which comprises the heavy and light chain amino acid sequences shown in Figure 6 of WO 2015026634 The amino acid sequence of pembrolizumab is described in WO2008156712 ( light chain CDRs SEQ ID NOS:15, 16 and 17 and heavy chain CDRs SEQ ID NOS: 18, 19 and 20)., In an embodiment of the invention the PD-1 antibody is nivolumab (BMS-936558, MDX 1106; WHO Drug Information, Vol. 27, No. 1, pages 68-69 (2013), W02006/121168 amino acid sequences shown in WO 2015026634). In an embodiment of the invention the PD-1 antibody is; pidilizumab (CT-011, also known as hBAT or hBAT-1; amino acid sequence see W02003/099196; WO 2009/101611, Friedl et al.; Neuro Oncol (2014) 16 (suppl 5): vlll-vll2.). In an embodiment of the invention the PD-1 antibody is MEDI- 0680 (AMP-514, WO2010/027423, WO2010/027827, WO2010/027828, Hamid O. et al; J Clin Oncol 33, 2015 (suppl; abstr TPS3087). In an embodiment of the invention the PD-1 antibody is PDR001 (Naing A. et al; J Clin Oncol 34, 2016 (suppl; abstr 3060). In an embodiment of the invention the PD-1 antibody is REGN2810 (Papadopoulos KPet al; J Clin Oncol 34, 2016 (suppl; abstr 3024). In an embodiment of the invention the PD-1 antibody is lambrolizumab (W02008/156712). In an embodiment of the invention the PD-1 antibody is h409Al 1, h409A16 or h409A17, which are described in WO2008/156712. The dosage of such anti-PD-1 antibody is performed according to the state of the art and described in the respective prescribing informations. E.g. Keytruda® is administered usually in a concentration of 2mg/kg body weight every three weeks (http://ec.europa.eu/heai th/documents).

The term “anti-PD-Ll antibody” as used herein relates to an antibody specifically binding to human PD-L1. Such antibodies are e.g. described in WO2015026634, WO2013/019906, W02010/077634 and US8383796. In an embodiment of the invention the PD-L1 antibody is MPDL3280A (atezolizumab, YW243.55.S70, WO2010/077634, McDermott DF. Et al, JCO March 10, 2016 vol. 34 no. 8 833-842). In an embodiment of the invention the PD-L1 antibody is MDX-1105 (BMS- 936559, W02007/005874, Patrick A. Ott PA et al, DOI: 10.1158/1078-0432, Clinical Cancer Research-13-0143). In an embodiment of the invention the PD-L1 antibody is MEDI4736 (durvalumab, WO 2016/040238 Gilbert J. et al, Journal for ImmunoTherapy of Cancer 20153(Suppl 2):P152). In an embodiment of the invention the PD-L1 antibody is MSB001071 8C (avelumab, Disis ML. et al., Journal of Clinical Oncology, Vol 33, No 15_suppl (May 20 Supplement), 2015: 5509). In an embodiment of the invention the PD-L1 antibody is the anti-PD- L1 antibody comprising a VH sequence of SEQ ID NO: 16 and a VL sequence of SEQ ID NO: 17 as described in W02016007235. The dosage of such anti-PD-Ll antibody is performed according to the state of the art and described in the respective prescribing informations. E.g. atezolizumab is administered usually in a concentration of 1200 mg as an intravenous infusion over 60 minutes every 3 weeks (¾vww. accessdata. fda. gov).

The term "gamma secretase" as used herein refers to any protein or protein complex that exhibits gamma secretase activities including binding to a substrate having a gamma secretase cleavage sequence, and catalyzing the cleavage of the gamma secretase cleavage sequence, at a gamma secretase cleavage site, to produce substrate cleavage products. In one embodiment, gamma secretase is a protein complex comprising one or more of the following subunits: presenilin, nicastrin, gamma-secretase subunit APH-1, and gamma-secretase subunit PEN-2.

The term "gamma secretase inhibitor" or "GSI" as used herein refers to any molecule capable of inhibiting or reducing expression and/or function of gamma secretase. In certain embodiment, the GSI reduces expression and/or function of a subunit of gamma secretase (e.g., presenilin, nicastrin, APH-1, or PEN-2). Any form of a "gamma secretase inhibitor" such as a salt, a co-crystal, a crystalline form, a pro-drug, etc., is included within this term. In some embodiments, the GSI is selected from an antibody or antigen-binding fragment, a small molecule, a protein or peptide and a nucleic acid.

The above embodiments are to be understood as illustrative examples. Further embodiments are envisaged. It is to be understood that any feature described in relation to any one embodiment may be used alone, or in combination with other features described, and may also be used in combination with one or more features of any other of the embodiments, or any combination of any other of the embodiments. Furthermore, equivalents and modifications not described above may also be employed without departing from the scope of the invention, which is defined in the accompanying claims.

In the context of the present invention other examples and variations of the antibodies and methods described herein will be apparent to a person of skill in the art. Other examples and variations are within the scope of the invention, as set out in the appended claims.

All documents cited herein are each entirely incorporated by reference herein, including all data, tables, figures, and text presented in the cited documents.

Table 4A: Antibody sequences

Remark: SEQ ID NO: 20 and SEQ ID NO: 33 are identical

Table 4B: Antibody sequences (short list)

Table 5A: Additional constructs

Table 5B: Additional constructs

Examples

The examples in this section are offered by way of illustration, and not by way of limitation.

The canonical expression pattern of BCMA begins on germinal center B-cells and becomes maximally expressed on mature cells such as plasma cells. BCMA is known to be highly expressed on multiple myeloma (MM) cells, the malignant counterpart to plasma cells. We sought to determine the expression of BCMA in lymphomas derived from earlier B-cell lineages. BCMA surface expression varies among a range of lymphoma cell lines, and most cell lines showed significantly reduced BCMA expression over MM, e.g. as low as 43 molecules per cell ((see Examples 1 and 4). Yet surprisingly, the lymphoma cell lines had sufficient BCMA surface expression for T cell cytotoxicity using the multispecific antibodies of the invention (see Example

3)·

Example 1: Quantification of BCMA cell surface expression by flow cytometry We first sought to directly quantitate cell surface expression of BCMA utilizing a flow cytometry system based on a logarithmic dilution of phycoerythrin beads of a known quantity. In a panel of 43 lymphoma cell lines from a range of cellular origins, including cell lines of DLBCL, transformed FL (RL and DOHH2) and MCL (JEKO-1), we determined that BCMA surface expression varies among NHL cell lines, ranging from 43 to 17,048 molecules per cell (median, 420) (FIG. 4). An isogenic pair of K562 that is null for BCMA expression and K562 constitutively overexpressing BCMA (K562-BCMA) (15,866 molecules/cell) served as negative and positive controls, respectively. The MM cell line H929 was profded to serve as an additional control with a BCMA expression level of 7,065 molecules/cell. BCMA surface expression is much lower than the MM cell line for nearly all of the tested lymphoma cell lines.

Example 2: Quantification of BCMA mRNA expression by PCR and determining correlation with BCMA cell surface expression

Next, utilizing quantitative PCR we found that relative BCMA mRNA expression in the lymphoma cell lines ranged from 0.001 to 0.17-fold when normalized to the H929 MM cell line. Furthermore, we found that in the lymphoma cells there is a poor correlation between BCMA cell surface expression (mean fluorescent intensity) and BCMA mRNA expression (r 2 , 0.3377; p<0.001) (FIG.

5)·

Example 3: Measuring T-cell mediated cytotoxicity after administration of CC-93269 and determining correlation with BCMA cell surface expression

We next examined whether there was any correlation between BCMA surface expression and T- cell mediated cytotoxicity after administration of CC-93269, a bispecific antibody that specifically binds to BCMA and CD3, in a co-culture assay.

We selected 11 lymphoma cell lines from Example 1 having a BCMA surface expression ranging from 43 molecules to 17,048 molecules per cells and incubated them in a co-culture system with 1:5 target: effector cells (isolated CD3+ T-cells) with CC-93269 (0-200 ng/ml) for 72 hours. Results were normalized to a control bispecific antibody where the two binding domains for BCMA was altered to target HEL (hen egg lysozyme), which demonstrated no activity against any of the cell lines profiled at a defined dose of 200 ng/ml.

Significant apoptosis as measured by annexin V and ToPro-3 staining of CFSE-positive target cells was observed in 10 of the 11 lymphoma cell lines, exhibiting an IC50 of 0.1 to 38 ng/ml. The K562 isogenic pair of Example 1 were profiled as controls. The K562-BCMA cell line exhibited an IC50 of 0.5 ng/ml and no activity was observed against the parental K562 cell line (FIG. 6). The results demonstrate that the lymphoma cell lines are sensitive to CC-93269-mediated cytotoxicity at levels of at least 200 ng/ml, irrespective of the levels of BCMA surface expression. No association was found between CC-93269 activity and the lymphoma cell of origin (/. e. arising from germinal center B cells or activated B cells). These data highlight the potential for the treatment of lymphoma patients with a T-cell bispecific targeting BCMA.

Example 4: Quantification of BCMA cell surface expression by immunohistochemistry

To determine the expression of BCMA in lymphoma biopsy samples, we developed a novel monoclonal BCMA immunohistochemistry antibody (clone G12). The antibody and IHC staining protocol were validated to have good on-target specificity and dynamic range in both cell lines and primary tissues, including DLBCL and MM biopsies, with staining observed in both the golgi and on the plasma membrane.

DLBCL (front line and relapsed/refractory) and follicular lymphoma (front line) biopsy samples were tested for BCMA cell surface expression using the above IHC protocol. A range of BCMA expression was detected in the DLBCL and FL biopsies, with intensities ranging from weakly positive (1+) to strongly positive (3+) (see FIG. 7 for representative images).

A cohort of frontline DLBCL biopsies, a second cohort of R/R DLBCL biopsies and a third cohort of frontline follicular lymphoma biopsies were analysed. PAX5 expression within tumor sections of each cohort was analysed to detect malignant B cells, as Pax5 is a reliable B-cell marker (FIG. 8A). IHC staining of BCMA revealed how many of the Pax5+ malignant B cells in each tumor section stain positive for BCMA (FIG. 8B). Of those BCMA positive cells, a range of staining intensities was observed (1 = light; 2 = medium; and 3 = high ), indicating that there is variation of BCMA cell surface expression within the Pax5+ malignant B cells (FIG. 8C).

To visualize the percentage of BCMA-positive tumor cells with each BCMA scoring intensity, we used a scatter plot of the two variables to exemplify the heterogeneity between BCMA positivity and scoring intensity in B-NHL tumors (FIG. 9).

Each cohort was analyzed to determine the percentage of tumor sections which are considered to be BCMA-expressing by the IHC protocol above, using a cut-off of 1% (FIG. 10A), 5% (FIG. 10B) and 1%, 26%, 51%, and 76% (FIG. IOC) of BCMA expression.

Our data demonstrate that BCMA is expressed on the cell surface of a broad panel of NHL cell lines and in DLBCL and follicular lymphoma lymph node biopsies. Additionally, the expression levels of BCMA in the cell line models was sufficient to elicit significant CC-93269-mediated T cell cytotoxicity. These data highlight the potential for the treatment of lymphoma patients with a T-cell bispecific targeting BCMA.

Example 5 : Evaluation of CC-93269 on follicular lymphoma samples

The efficacy of CC-93269 on primary lymph-node derived FL tumor samples was evaluated in vitro. FL samples (n = 4) were mechanically dissected, and single cell suspensions were obtained through mixing the tumor suspension in a strainer. The single cell suspensions were treated for 7 days with CC-93269 at a concentration of 0.1 pg/ml, 0.5 pg/ml or 1 pg/ml. Vehicle (phosphate buffered saline) and a CD20xCD3 bispecific antibody served as negative and positive controls, respectively. Following incubation, the B cells and T cells in the samples were identified by flow cytometry (using measurements of CD19+ for B cells, and of CD3+, CD4+ and CD8+ for T cells).

Treating FL tumor samples with CC-93269 at 1 pg/ml resulted in the B-cell count being decreased by 32-81% compared to the vehicle control (FIG. 11 A). CC-93269 treatment also resulted in expansion of CD4+ and CD8+ T-cells within the FL tumor samples (FIG. 11B). The positive control, CD20xCD3 bispecific antibody, also resulted in decreased B-cells (47-97%) and increased T-cells compared to the vehicle control.

EQUIVALENTS

Although the invention is described in detail with reference to specific embodiments thereof, it will be understood that variations which are functionally equivalent are within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.