STATEMENT OF PRIORITY

[0001] This application claims the benefit of U.S. Provisional Application Serial No. 63/398,804, filed August 17, 2022, the entire contents of which are incorporated by reference herein.

STATEMENT REGARDING ELECTRONIC FILING OF A SEQUENCE LISTING [0002] A Sequence Listing in XML text format, submitted under 37 C.F.R. § 1.821-1.834, entitled 1548-6WO_ST26.xml, 41,977 bytes in size, generated on August 14, 2023, and filed electronically, is provided in lieu of a paper copy. This Sequence Listing is hereby incorporated herein by reference into the specification for its disclosures.

FIELD OF THE INVENTION

[0003] The present invention relates to binding agents and compositions comprising the same. The invention further relates to polynucleotides encoding the binding agent and vectors and host cells comprising the same. The invention further relates to methods of using the binding agents to promote macrophage mediated antibody-dependent cellular cytotoxicity and antibody-dependent cellular phagocytosis of cancer cells and methods of treating cancers.

BACKGROUND

[0004] Antibodies are proteins that bind to a specific antigen. Monoclonal antibodies (mAbs) and mAb-based reagents approved for cancer therapy include several that are directed against antigens expressed on malignant B cells and plasma cells (CD19, CD20, CD22, CD30, CD38, CD52, CD79B, SLAMF7), epithelial cancer cells (EpCAM, EGFR, HER2, VEGFR2, nectin- 4), acute myeloid leukemia (CD33), cutaneous T-cell lymphoma (CCR4), neuroblastoma (GD2), and sarcoma (PDGFRA), as well as immune checkpoint targets (PD-1, PD-L1, CTLA- 4) (Gasser, 2016; Carter, 2018). A total of 42 antibody -based cancer therapies are currently FDA-approved and marketed. The efficacy of a therapeutic antibody for cancer can be influenced by a combination of mechanisms (Chiavenna, 2017). Antibody binding to an antigen selectively expressed on a cancer cell may produce anti-tumor effects by directly blocking the function of the antigen that promotes tumor cell growth or survival pathways. An antibody can also act as a bridge to bring together a tumor cell with an immune effector cell that can indirectly induce tumor cell destruction.

[0005] The properties of therapeutic antibodies can be modified to either enhance or suppress engagement with certain types of immune effector cells using a growing arsenal of glycoengineering and Fc engineering approaches or through the creation of bispecific or trispecific antibodies (Saxena, 2016; Rader, 2020). These tools can be utilized for the rational design of “antigen-effector matching” to create a personalized medicine approach for cancer therapy.

[0006] Antibody engineering strategies focused on improving the engagement of monocytes or natural killer (NK) cells include a vast collection of glycoengineered and Fc engineered variants that promote binding of the Fc portion of a therapeutic antibody to FcyRIIIA (CD16A), the only Fc receptor expressed on NK cells (Lazar, 2006). Although less common, several strategies have generated antibody variants with enhanced binding to macrophages, including a G236A Fc mutant that promotes binding to FcyRIIA (CD32A) (Richards, 2008) or a bispecific antibody that recruits macrophages via FcaRI (CD89) (Li, 2017). In contrast, it is desirable to engineer antibodies that do not bind Fc gamma receptors at all to avoid unwanted inflammatory responses to therapeutic antibodies and fusion proteins (Wilkinson, 2021).

[0007] Therapeutic antibodies can influence cancer progression through several mechanisms of action, including blocking the function of a target antigen or inhibiting ligand/receptor interactions. In addition, antibodies can also activate immune cells to induce tumor cell killing or engulfment, known as antibody-dependent cellular cytotoxicity (ADCC) or antibodydependent cellular phagocytosis (ADCP), respectively. Once an antibody binds to antigens on the surface of a target cell, Fc receptors on immune effector cells that recognize the antibody Fc domain mediate tumor cell killing. The effectiveness of this response is modulated by additional interactions, including the CD47 “don’t eat me” signal that some tumor cells can emit to evade macrophage phagocytosis. A variety of CD47-targeted agents have been developed that could be used in combination with antibody therapeutics to remove this brake on macrophage function.

[0008] Thus, there is a need for new compositions, and methods of using such compositions, to treat cancers, including in particular neuroblastoma and other GD2-expressing cancers.

SUMMARY OF THE DISCLOSURE

[0009] The present invention provides compositions and methods for engaging the tumor associated macrophage as the appropriate immune effector cell to effectively mediate one or both of two distinct killing mechanisms; antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) against cancer cells that express the cell surface marker GD2 (e.g., neuroblastoma, breast cancer, melanoma, small cell lung cancer).

[0010] While many antibodies against tumor antigens kill tumor cells via ADCC which is mediated by macrophages or NK cells, certain antibodies only are only able to engage macrophages to induced ADCC. This has been demonstrated with a chimeric antibody (ABT- 101) targeted to avP3 (WO 2021/216956, incorporated by reference herein in its entirety). Further, the cell death does not involve antibody-dependent cellular phagocytosis (ADCP) or direct killing via the antibody alone. The present invention is based on the development of binding agents that can mediate ADCC by selectively engaging myeloid-derived cells found in tumors and targeting them to the antigen GD-2 on the surface of expressing cancer cells (e.g., an IgG4 domain to engage macrophage CD64/FcyRl on macrophages and GD2 recognition).

[0011] A binding agent that promotes ADCC exclusively will facilitate some level of tumor cell killing but will not induce ADCP. To achieve optimal tumor killing a single binding agent would ideally mediate both ADCC and ADCP. However, a binding agent that promotes ADCP will not be able to engulf or kill CD47-positive tumor cells that emit the “don’t eat me” signal that blocks phagocytosis by macrophages. By combining a binding agent structure capable of ADCC-inducing effects (e.g., an IgG4 domain to engage macrophage CD64/FcyRl on macrophages and GD2 recognition) with a CD47 blocking agent capable of ADCP-inducing effects, more efficient cancer cell killing may be achieved, including CD47-positive tumor cells. Additionally, the preferential binding of CD64/FcyRl relative to CD 16 and CD32 on the macrophage facilitates maximal effector cell function to optimize macrophage-dependent killing. Thus, a binding agent that targets GD2 and CD47 on a tumor cell while simultaneously binding to CD64/FcyRl preferentially over CD 16 and CD32 on the macrophage will promote the most complete anti-tumor activity of GD2-expressing tumors.

[0012] Thus, one aspect of the invention relates to a binding agent e.g., an antibody fusion protein) comprising a first domain that specifically binds to GD2and a second domain that preferentially binds CD64/FcyRl on macrophages, wherein the binding agent mediates antibody-directed cellular cytotoxicity (ADCC) by selectively the macrophage that accumulates in mesenchymal tumors, and compositions or pharmaceutical compositions comprising the binding agents. In some embodiments, the binding agent further comprises a third domain that is a CD47 blocking agent, wherein the binding agent mediates ADCC and antibody-dependent cellular phagocytosis (ADCP). [0013] Another aspect of the invention relates to a polynucleotide encoding the binding agent of the invention and vectors and host cells comprising the polynucleotide.

[0014] An additional aspect of the invention relates to a method of killing a cancer cell expressing GD2 or expressing GD2 and CD47, comprising contacting the cell with an effective amount of the binding agent of the invention.

[0015] A further aspect of the invention relates to a method of targeting a macrophage to a cancer cell expressing GD2 or expressing GD2 and CD47, comprising contacting the cancer cell and the macrophage with an effective amount of the binding agent of the invention.

[0016] An additional aspect of the invention relates to a method of inducing macrophagedependent antibody-dependent cellular cytotoxicity (ADCC) and/or antibody-dependent cellular phagocytosis (ADCP) against a cancer expressing GD2 or expressing GD2 and CD47 in a subject in need thereof, comprising administering a therapeutically effective amount of the binding agent or the pharmaceutical composition of the invention to the subject, thereby inducing macrophage-dependent ADCC and/or ADCP.

[0017] A further aspect of the invention relates to a method of treating a cancer expressing GD2 or expressing GD2 and CD47 in a subject in need thereof, comprising administering a therapeutically effective amount of the binding agent or the pharmaceutical composition of the invention to the subject, wherein the administering induces ADCC and/or ADCP of the cancer, thereby treating the cancer.

[0018] Another aspect of the invention relates to a method of treating a cancer in a subject in need thereof, comprising the steps of: a) selecting a subject having cancer cells that are enriched for GD2 or GD2 and CD47 and enriched for macrophages; and b) administering a therapeutically effective amount of the binding agent or the pharmaceutical composition of the invention to the subject, wherein the administering induces ADCC and/or ADCP of the cancer, thereby treating the cancer.

[0019] These and other aspects of the invention are set forth in more detail in the description of the invention below.

BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 shows that, among immune effector cells that are known to induce ADCC/ADCP, macrophage markers were most enriched in neuroblastoma patient tissues. Immune cell markers were compared in neuroblastoma patient TCGA data. *P<0.05 compared to macrophage using TTEST. Error bars indicate standard errors. NK, natural killer; DC, dendritic cell.

[0021] Figure 2 shows h!gG4-Anti-GD2 maintains affinity for GD2 on neuroblastoma cells. SK-N-AS neuroblastoma cells were stained with Anti-GD2 (dinutuximab) or h!gG4-Anti- GD2, and flow cytometry was performed. Dotted line, secondary antibody only; solid line, primary + secondary antibodies.

[0022] Figure 3 shows neuroblastoma cells highly express CD47. Levels of CD47 were analyzed in SHEP neuroblastoma cells using flow cytometry. Dotted line, isotype control; solid line, anti-CD47.

[0023] Figure 4 shows h!gG4 anti-GD2 antibody induces macrophage-mediated neuroblastoma cell elimination. SK-N-AS (neuroblastoma, luciferase+, GD2+) cells were incubated with PBS or h!gG4 anti-GD2 (10 pg/mL) at the E:T ratio of 10: 1 with human macrophages for 24 hr. SK-N-AS cell death was measured by reading luminescence signals. Error bars indicate standard deviation of technical replicates. *P<0.05 compared to PBS.

[0024] Figure 5 shows anti-GD2 antibody (dinutuximab) effectively kills target cells through an NK-cell mediated mechanism but not h!gG4-Anti-GD2. Target cells (SHEP-luc, GD2+, luciferase+) were incubated with the indicated antibody at 1 pg/mL with human NK cells at the E:T ratios of 10: 1 for 24 hr. Percent antibody-dependent target cell death was analyzed using a luminometer. Error bar indicates standard deviation for technical replicates. *P<0.05 vs control.

[0025] Figure 6 shows h!gG4 anti-GD2 antibody induces human macrophage-mediated ADCC. Target cells (SHEP-luc, GD2+, luciferase+) were incubated with the indicated antibody at the indicated concentration with human macrophages at the E:T ratios of 10: 1 for 24 hr. Percent antibody-dependent target cell death was analyzed using a luminometer. Error bar indicates standard deviation for technical replicates. *P<0.05 vs control.

[0026] Figure 7 shows h!gG4 anti-GD2 antibody induces mouse macrophage-mediated ADCC. Target cells (SHEP-luc, GD2+, luciferase+) were incubated with the indicated antibody at the indicated concentration with human macrophages at the E:T ratios of 10: 1 for 24 hr. Percent antibody-dependent target cell death was analyzed using a luminometer. Error bar indicates standard deviation for technical replicates. *P<0.05 vs control.

[0027] Figure 8 shows h!gG4 anti-GD2 antibody kills HCC1395 and Hs578T human triple negative breast cancer cells to a greater extent than the anti-GD2 antibody dinutuximab. DETAILED DESCRIPTION

[0028] The present invention is explained in greater detail below. This description is not intended to be a detailed catalog of all the different ways in which the invention may be implemented, or all the features that may be added to the instant invention. For example, features illustrated with respect to one embodiment may be incorporated into other embodiments, and features illustrated with respect to a particular embodiment may be deleted from that embodiment. In addition, numerous variations and additions to the various embodiments suggested herein will be apparent to those skilled in the art in light of the instant disclosure which do not depart from the instant invention. Hence, the following specification is intended to illustrate some particular embodiments of the invention, and not to exhaustively specify all permutations, combinations, and variations thereof.

[0029] Unless the context indicates otherwise, it is specifically intended that the various features of the invention described herein can be used in any combination. Moreover, the present invention also contemplates that in some embodiments of the invention, any feature or combination of features set forth herein can be excluded or omitted. To illustrate, if the specification states that a complex comprises components A, B and C, it is specifically intended that any of A, B or C, or a combination thereof, can be omitted and disclaimed singularly or in any combination.

[0030] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0031] Except as otherwise indicated, standard methods known to those skilled in the art may be used for production of recombinant and synthetic polypeptides, antibodies, or antigenbinding fragments thereof, manipulation of nucleic acid sequences, and production of transformed cells. Such techniques are known to those skilled in the art. See, e.g., SAMBROOK et al., MOLECULAR CLONING: A LABORATORY MANUAL 4th Ed. (Cold Spring Harbor, N.Y., 2012); F. M. AUSUBEL et al. CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Green Publishing Associates, Inc. and John Wiley & Sons, Inc., New York).

[0032] All publications, patent applications, patents, nucleotide sequences, amino acid sequences and other references mentioned herein are incorporated by reference in their entirety. Definitions

[0033] As used in the description of the invention and the appended claims, the singular forms “a,” “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0034] As used herein, “and/or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”).

[0035] Moreover, the present invention also contemplates that in some embodiments of the invention, any feature or combination of features set forth herein can be excluded or omitted.

[0036] Furthermore, the term “about,” as used herein when referring to a measurable value such as an amount of a compound or agent of this invention, dose, time, temperature, and the like, is meant to encompass variations of ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of the specified amount.

[0037] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about”. Accordingly, unless indicated to the contrary, the numerical parameters set forth in this specification and claims are approximations that can vary depending upon the desired properties sought to be obtained by the presently-disclosed subject matter.

[0038] As used herein, ranges can be expressed as from “about” one particular value, and/or to “about” another particular value. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. It is also understood that each unit between two particular units is also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0039] The transitional phrase “consisting essentially of’ means that the scope of a claim is to be interpreted to encompass the specified materials or steps recited in the claim, and those that do not materially affect the basic and novel characteristic(s) of the claimed invention.

[0040] The term “consists essentially of’ (and grammatical variants), as applied to a polynucleotide or polypeptide sequence of this invention, means a polynucleotide or polypeptide that consists of both the recited sequence (e.g., SEQ ID NO) and a total of ten or less (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) additional nucleotides or amino acids on the 5’ and/or 3’ or N-terminal and/or C-terminal ends of the recited sequence or between the two ends (e.g., between domains) such that the function of the polynucleotide or polypeptide is not materially altered. The total of ten or less additional nucleotides or amino acids includes the total number of additional nucleotides or amino acids added together.

[0041] As used herein, the term “polypeptide” encompasses both peptides and proteins, unless indicated otherwise.

[0042] The term “chimeric” refers to a molecule having two or more portions that are not naturally found together in the same molecule.

[0043] A “nucleic acid” or “nucleotide sequence” or “polynucleotide” is a sequence of nucleotide bases, and may be RNA, DNA or DNA-RNA hybrid sequences (including both naturally occurring and non-naturally occurring nucleotide) but is preferably either single or double stranded DNA sequences.

[0044] As used herein, the term “isolated” means a molecule, e.g., a protein, polynucleotide, or cell, separated or substantially free from at least some of the other components of the naturally occurring organism or virus, for example, the cell structural components or other polypeptides or nucleic acids commonly found associated with the molecule. The term also encompasses molecules that have been prepared synthetically.

[0045] By the terms “treat,” “treating,” or “treatment of’ (or grammatically equivalent terms) it is meant that the severity of the subject's condition is reduced or at least partially improved or ameliorated and/or that some alleviation, mitigation or decrease in at least one clinical symptom is achieved and/or there is a delay in the progression of the condition.

[0046] As used herein, the terms “prevent,” “prevents,” or “prevention” and “inhibit,” “inhibits,” or “inhibition” (and grammatical equivalents thereof) are not meant to imply complete abolition of disease and encompasses any type of prophylactic treatment that reduces the incidence of the condition, delays the onset of the condition, and/or reduces the symptoms associated with the condition after onset.

[0047] An “effective,” “ prophy lactically effective,” or “therapeutically effective” amount as used herein is an amount that is sufficient to provide some improvement or benefit to the subject. Alternatively stated, an “effective,” “prophylactically effective,” or “therapeutically effective” amount is an amount that will provide some delay, alleviation, mitigation, or decrease in at least one clinical symptom in the subject. Those skilled in the art will appreciate that the effects need not be complete or curative, as long as some benefit is provided to the subject.

[0048] As used herein, the term “bind specifically” or “specifically binds” in reference to a binding agent of the invention means that the agent will bind with an epitope (including one or more epitopes) of a target, but does not substantially bind to other unrelated epitopes or molecules. In certain embodiments, the term refers to an agent that exhibits at least about 60% binding, e.g., at least about 70%, 80%, 90%, or 95% binding, to the target epitope relative to binding to other unrelated epitopes or molecules.

Binding agents

[0049] A first aspect of the invention relates to a binding agent comprising a first domain that specifically binds to GD2and a second domain that preferentially binds CD64 relative to CD 16 and CD32 on macrophages, wherein the binding agent mediates antibody-directed cellular cytotoxicity (ADCC) by engaging a macrophage that accumulates in tumors. In some embodiments, the binding agent further comprises a third domain that is a CD47 blocking agent, wherein the binding agent mediates ADCC and antibody-dependent cellular phagocytosis (ADCP).

[0050] The binding agent may be any structure that is capable of binding to an antigen on a cancer cell and engaging a myeloid-derived cell to mediate ADCC and/or ADCP. In some embodiments, the binding agent is an antibody or an antigen-binding fragment thereof. In some embodiments, one or more portions of the binding agent are composed of antibody fragments. In some embodiments, one or more domains of the binding agent is a non-immunoglobulin scaffold, an aptamer, a small molecule (e.g., a receptor ligand), or other binding moiety. In some embodiments, the binding agent is a fusion protein in which the first and second domain are fused, the second and third domain are fused, or the first second, and third domain are fused. The domains may be fused together directly or through a linker. The linker may be 1 to about 20 amino acid residues, e.g., composed of glycine and serine residues, e.g., (GGGGS)n, wherein n is 1-5.

[0051] In certain embodiments, the first domain of the binding agent is an antibody domain. In certain embodiments, the second domain of the binding agent is an antibody domain. In certain embodiments, the third domain of the binding agent is an antibody domain. In some embodiments, the first and third domains are antibody domains. In some embodiments, all of the domains are antibody domains. In some embodiments, the first domain is a humanized or human antibody domain. In some embodiments, the second domain is a humanized or human antibody domain. In some embodiments, the third domain is a humanized or human antibody domain. In some embodiments, the first domain and the third domain are humanized or human antibody domains. In some embodiments, all of the domains are humanized or human antibody domains. [0052] In certain embodiments, the first domain comprises, consists essentially of, or consists of a Fab domain of an antibody. The Fab domain may be from any antibody isotype. In some embodiments, the first domain comprises a Fab domain of an IgG antibody, e.g., an IgGl or IgG4 antibody. In some embodiments, the first domain comprises the Fab domain of dinutuximab or a sequence at least 90% identical thereto, e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical thereto. The heavy and light chain sequences of dinutuximab are disclosed in SEQ ID NOS: 1 and 2, respectively.

[0053] The second domain of the binding agent preferably engages one or more types of myeloid-derived cells. In some embodiments, the second domain predominately engages one type of myeloid-derived cells, e.g., macrophages or dendritic cells or granulocytes, such as a neutrophils, basophils, eosinophils, or mast cells. In some embodiments, the second domain predominately engages macrophages. “Predominantly engage,” as used herein, refers to engaging at least 80% of the target cell type, e.g., macrophages, relative to other cell types, e.g., at least 85%, 90%, or 95%.

[0054] In certain embodiments, the second domain does not significantly engage natural killer (NK) cells. In certain embodiments, the second domain does not significantly engage one or more types of lymphocytes, e.g., NK cells, B cells, or T cells. “Does not significantly engage,” as used herein, refers to less than 30% of the total engaged cells being the indicated cell type, e.g., less than 25%, 20%, 15%, 10%, or 5%.

[0055] In some embodiments, the second domain specifically binds a protein on the surface of the myeloid-derived cell. The protein is one that can mediate ADCC when engaged. In some embodiments, the protein is not present or only present at low levels on other cell types, e.g., natural killer cells. In some embodiments, the second domain specifically binds to an Fc- gamma receptor. In some embodiments, the second domain specifically binds Fc-gamma receptor I (FcyRI, CD64) or preferentially binds CD64 relative to CD16 and CD32. The term “preferentially binds”, as used herein, refers to a binding affinity of the second domain to CD64 that is at least 5-fold higher than the binding affinity to CD 16 and CD32, e.g., at least 10-fold, 50-fold, or 100-fold higher.

[0056] In certain embodiments, the second domain comprises, consists essentially of, or consists of an Fc domain of an antibody. The Fc domain may be from any antibody isotype. In some embodiments, the second domain comprises an Fc domain of an IgG antibody, e.g., an IgG4 antibody. In some embodiments, the second domain comprises an Fc domain of an IgA or IgE antibody. In certain embodiments, the second domain further comprises a hinge domain of an antibody. In some embodiments, the second domain comprises an antibody or an antigen-binding fragment thereof that specifically binds CD64.

[0057] The third domain of the binding agent is a CD47 blocking agent, i.e., an agent that inhibits the “don’t eat me” signal of CD47. In some embodiments, the third domain specifically binds CD47. In certain embodiments, the third domain is an antibody or an antibody domain that specifically binds CD47. CD47-blocking monoclonal antibodies are known in the art (e.g., Hu5F9-G4, CC-90002, Ti-061, or SRF231). In other embodiments, the third domain is a CD47 ligand or a fragment or derivative thereof that specifically binds CD47 and inhibits CD47 signaling.

[0058] In certain embodiments, the CD47 ligand is SIRPa or a fragment or derivative thereof. The SIRPa fragments or derivatives may comprise the amino acid sequence of a wild-type SIRPa protein or a modified amino acid sequence. Numerous examples of inhibitory SIRPa fragments or derivatives are known in the art. In some embodiments, the SIRPa or a fragment or derivative thereof has the same affinity to CD47 as a wild-type SIRPa protein, increased affinity, or decreased affinity. Examples include, without limitation, the N-terminal Ig-like domain (e.g., residues 1-118) of fragments or derivatives thereof. See, e.g., Patent Nos. US 9,944,911, US 10,487,150, WO 2017/081101, WO 2020/029982, US 10,696,730, US 11,021,694, US 2018/0195054, EP 3872086, US 2021/0070838, EP 3128005, US 2021/0206829, US 2018/0311348, each incorporated by reference herein for their disclosure of SIRPa fragments or derivatives.

[0059] In some embodiments, the SIRPa fragment comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 19 (the wild-type N-terminal Ig-like domain) or a sequence at least 90% identical thereto, e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical thereto. In other embodiments, the SIRPa derivative comprises the amino acid sequence of SEQ ID NO:21 (a truncated fragment of the N-terminal Ig-like domain) or a sequence at least 90% identical thereto, e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical thereto.

[0060] In other embodiments, the SIRPa derivative comprises the amino acid sequence of SEQ ID NO:20 (the N-terminal Ig-like domain in which the three glutamic acid residues at the N-terminus have been substituted with 4 aspartic acid residues) or a sequence at least 90% identical thereto, e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical thereto.

[0061] In some embodiments, the SIRPa derivative comprises a transglutaminase conjugation site (LLQG; SEQ ID NO:27). These sites can be used to make conjugates of the binding agent, e.g., antibody-drug conjugates (see, e.g., Strop, 2013). In some embodiments, the SIRPa derivative comprises the amino acid sequence of one of SEQ ID NO:22 (transglutaminase conjugation site in one of the first loops), SEQ ID NO:23 (transglutaminase conjugation site in one of the first loops), SEQ ID NO:24 (transglutaminase conjugation site in the EF loop to replace the sequence SNITPADA (SEQ ID NO:28)), or a sequence at least 90% identical to one of SEQ ID NOS: 22-24, e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical thereto.

[0062] In some embodiments, the SIRPa derivative comprises mutations to alter one or more of the salt bridges that from during SIRPa-CD47 interaction. By weakening the binding between the two proteins, these mutations may permit SIRPa-CD47 interaction only when the SIRPa is brought into the proximity of CD47 by the binding of the binding agent. In some embodiments, the mutation is R69A and/or D100A. In some embodiments, the SIRPa derivative comprises the amino acid sequence of one of SEQ ID NO:25 (R69A mutation) or SEQ ID NO:26 (D100A mutation), or a sequence at least 90% identical to one of SEQ ID NOS: 25-26, e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical thereto.

[0063] In certain embodiments, the SIRPa derivative may comprise any combination of the described modifications, e.g., truncation and/or transglutaminase conjugation site(s) and/or salt bridge mutation(s).

[0064] One or more of the CD47 blocking agents may be included in the binding agent, e.g., 2, 3, 4, or more. The one or more CD47 blocking agents may be linked to each other to form a chain. Each CD47 blocking agent may be the same or different from other CD47 blocking agents that are part of the binding agent. The CD47 blocking agent(s) may be linked to the first domain, the second domain, or both. The CD47 blocking agent may be linked to the light chain fragment, the heavy chain fragment, or both of the first domain and/or the second domain. [0065] The one or more CD47 blocking agents may be linked to each other and/or to the first and/or second domains by a linker. The linker may be 1 to about 20 amino acid residues, e.g., composed of glycine and serine residues, e.g., (GGGGS)n, wherein n is 1-5.

[0066] In some embodiments, the binding agent comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:3 or a sequence at least 90% identical thereto, e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical thereto. In some embodiments, the binding agent comprises a light chain comprising the amino acid sequence of SEQ ID NO:4 or a sequence at least 90% identical thereto, e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical thereto. In some embodiments, the binding agent comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:3 or a sequence at least 90% identical thereto, e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical thereto, and a light chain comprising the amino acid sequence of SEQ ID NO:4 or a sequence at least 90% identical thereto, e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical thereto.

[0067] In some embodiments, the binding agent comprises: a heavy chain comprising the amino acid sequence of SEQ ID NO:5 or a sequence at least 90% identical thereto, e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical thereto, and a light chain comprising the amino acid sequence of SEQ ID NO:6 or a sequence at least 90% identical thereto, e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical thereto; a heavy chain comprising the amino acid sequence of SEQ ID NO:7 or a sequence at least 90% identical thereto, e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical thereto, and a light chain comprising the amino acid sequence of SEQ ID NO:8 or a sequence at least 90% identical thereto, e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical thereto; a heavy chain comprising the amino acid sequence of SEQ ID NO:9 or a sequence at least 90% identical thereto, e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical thereto, and a light chain comprising the amino acid sequence of SEQ ID NO: 10 or a sequence at least 90% identical thereto, e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical thereto; a heavy chain comprising the amino acid sequence of SEQ ID NO: 11 or a sequence at least 90% identical thereto, e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical thereto, and a light chain comprising the amino acid sequence of SEQ ID NO: 12 or a sequence at least 90% identical thereto, e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical thereto; a heavy chain comprising the amino acid sequence of SEQ ID NO: 13 or a sequence at least 90% identical thereto, e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical thereto, and a light chain comprising the amino acid sequence of SEQ ID NO: 14 or a sequence at least 90% identical thereto, e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical thereto; a heavy chain comprising the amino acid sequence of SEQ ID NO: 15 or a sequence at least 90% identical, e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical thereto, thereto and a light chain comprising the amino acid sequence of SEQ ID NO: 16 or a sequence at least 90% identical thereto, e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical thereto; or a heavy chain comprising the amino acid sequence of SEQ ID NO: 17 or a sequence at least 90% identical thereto, e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical thereto, and a light chain comprising the amino acid sequence of SEQ ID NO: 18 or a sequence at least 90% identical thereto, e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical thereto.

[0068] In some embodiments, the binding agent of the invention may be covalently or non- covalently linked to one or more other binding agents that are the same or different. In some embodiments, the binding agent of the invention may be linked to a binding agent that targets avP3 (e.g., a binding agent as described in WO 2021/216956). In certain embodiments, the two linked binding agents comprise the amino acid sequences of SEQ ID NOS:29-32 or SEQ ID NOS:33-36 or a sequence at least 90% identical thereto, e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical thereto.

[0069] The binding agent may include sequence modifications that are known to enhance the characteristics of an antibody, e.g., stability, or alter the binding of the antibody to Fc-gamma receptors. In some embodiments, the amino acid sequence of the binding agent comprises a S228P (Eu numbering system) mutation in the hinge region. In some embodiments, the amino acid sequence comprises a mutation selected from: a) S239D/A330L/I332E; b) I332E; c) G236A/S239D/I332E; d) G236A; e) N297A/E382V/M428I; f) M252Y/S254T/T256E; g) Q295R/L328 W/A330 V/P331 A/I332 Y/E382 V/M428I; h) L234A/L235A/P329G; i) M428L/N434S; j) L234A/L235A/P331S; k) L234A/L235A/P329G/M252Y/S254T/T256E; l) S298A/E333A/K334/A; m) S239D/I332E; n) G236A/S239D/A330L/I332E; o) S239D/I332E/G236A; p) L234Y/G236W/S298A; q) F243L/R292P/Y300L/V305I/P396L; r) K326W/E333S; s) K326A/E333A; t) K326M/E333S; u) C221D/D222C; v) S267E/H268F/S324W; w) H268F/S324W; x) E345R y) R435H; z) N434A; aa) M252Y/S254T/T256E; ab) M428L/N434S; ac) T252L/T/253S/T254F; ad) E294delta/T307P/N434Y; ae) T256N/A378V/S383N/N434Y; af) E294delta ag) L235E; ah) L234A/L235A; ai) S228P/L235E; aj) P331S/L234E/L225F; ak) D265A; al) G237A; am) E318 A; an) E233P; ao) G236R/L328R; ap) H268Q/V309L/A330S/P331S; aq) L234 A/L235 A/G237 A/P238 S/H268 A/A330 S/P331 S ; ar) A330L; as) D270A; at) K322A; au) P329A; av) P331A; aw V264A; ax) F241A; ay) N297A or G or N az) S228P/F234A/L235A; or ba) any combination of a) to az);

(Eu numbering system) with or without the S228P mutation.

[0070] The following discussion is presented as a general overview of the techniques available for the production of antibodies; however, one of skill in the art will recognize that many variations upon the following methods are known.

[0071] The term “antibody” or “antibodies” as used herein refers to all types of immunoglobulins, including IgG, IgM, IgA, IgD, and IgE. The antibody can be monoclonal, oligoclonal, or polyclonal and can be of any species of origin, including (for example) mouse, rat, hamster, rabbit, horse, cow, goat, sheep, pig, camel, monkey, or human, or can be a chimeric or humanized antibody. See, e.g., Walker et al., Molec. Immunol. 26:403 (1989). The antibodies can be recombinant monoclonal antibodies produced according to the methods disclosed in U.S. Pat. No. 4,474,893 or U.S. Pat. No. 4,816,567. The antibodies can also be chemically constructed according to the method disclosed in U.S. Pat. No. 4,676,980.

[0072] Antibody fragments included within the scope of the present invention include, for example, Fab, Fab', F(ab)2, and Fv fragments; domain antibodies, diabodies; vaccibodies, linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. Such fragments can be produced by known techniques. For example, F(ab')2 fragments can be produced by pepsin digestion of the antibody molecule, and Fab fragments can be generated by reducing the disulfide bridges of the F(ab')2 fragments. Alternatively, Fab expression libraries can be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity (Huse et al., Science

(1989)). In some embodiments, the term “antibody fragment” as used herein may also include any protein construct that is capable of binding a target antigen.

[0073] Antibodies of the invention may be altered or mutated for compatibility with species other than the species in which the antibody was produced. For example, antibodies may be humanized or camelized. Humanized forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a complementarity determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity, and capacity. In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the framework (FR) regions (i.e., the sequences between the CDR regions) are those of a human immunoglobulin consensus sequence. The humanized antibody can be a superhumanized antibody where only two CDRs are non-human (US Patent No. 7,087,409). The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin (Jones et al., Nature 321 :522 (1986); Riechmann et al., Nature, 332:323 (1988); and Presta, Curr. Op. Struct. Biol. 2:593 (1992)).

[0074] Methods for humanizing non-human antibodies are well known in the art. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as “import” residues, which are typically taken from an “import” variable domain. Humanization can essentially be performed following the method of Winter and co-workers (Jones et al., Nature 321 :522 (1986); Riechmann et al., Nature 332:323 (1988); Verhoeyen et al., Science 239: 1534 (1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such “humanized” antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues (e.g., all of the CDRs or a portion thereof) and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

[0075] Human antibodies can also be produced using various techniques known in the art, including phage display libraries (Hoogenboom and Winter, J. Mol. Biol. 227:381 (1991); Marks et al., J. Mol. Biol. 222:581 (1991)). The techniques of Cole et al. and Boerner et al. are also available for the preparation of human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boerner et al., J. Immunol. 147:86 (1991)). Similarly, human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the following scientific publications: Marks et al., Bio/Technology 10:779 (1992); Lonberg et al., Nature 368:856 (1994); Morrison, Nature 368:812 (1994); Fishwild et al., Nature Biotechnol. 14:845 (1996); Neuberger, Nature Biotechnol. 14:826 (1996); Lonberg and Huszar, Intern. Rev. Immunol. 13:65 (1995).

[0076] Immunogens (antigens) are used to produce antibodies specifically reactive with target polypeptides. Recombinant or synthetic polypeptides and peptides, e.g., of at least 5 (e.g., at least 7 or 10) amino acids in length, or greater, are the preferred immunogens for the production of monoclonal or polyclonal antibodies. In one embodiment, an immunogenic polypeptide conjugate is also included as an immunogen. The peptides are used either in pure, partially pure, or impure form. Suitable polypeptides and epitopes for target pathogens and sperm are well known in the art. Polynucleotide and polypeptide sequences are available in public sequence databases such as GENBANK®/GENPEPT®. Large numbers of antibodies that specifically bind to target cancer cell antigens have been described in the art and can be used as starting material to prepare the antibodies of the present invention. Alternatively, new antibodies can be raised against target antigens using the techniques described herein and well known in the art.

[0077] Recombinant polypeptides are expressed in eukaryotic or prokaryotic cells and purified using standard techniques. The polypeptide, or a synthetic version thereof, is then injected into an animal capable of producing antibodies. Either monoclonal or polyclonal antibodies can be generated for subsequent use in immunoassays to measure the presence and quantity of the polypeptide.

[0078] Methods of producing polyclonal antibodies are known to those of skill in the art. In brief, an immunogen, e.g., a purified or synthetic peptide, a peptide coupled to an appropriate carrier (e.g., glutathione-S-transferase, keyhole limpet hemocyanin, etc.), or a peptide incorporated into an immunization vector such as a recombinant vaccinia virus is optionally mixed with an adjuvant and animals are immunized with the mixture. The animal's immune response to the immunogen preparation is monitored by taking test bleeds and determining the titer of reactivity to the peptide of interest. When appropriately high titers of antibody to the immunogen are obtained, blood is collected from the animal and antisera are prepared. Further fractionation of the antisera to enrich for antibodies reactive to the peptide is performed where desired. Antibodies, including binding fragments and single chain recombinant versions thereof, against the polypeptides are raised by immunizing animals, e.g., using immunogenic conjugates comprising a polypeptide covalently attached (conjugated) to a carrier protein as described above. Typically, the immunogen of interest is a polypeptide of at least about 10 amino acids, in another embodiment the polypeptide is at least about 20 amino acids in length, and in another embodiment, the fragment is at least about 30 amino acids in length. The immunogenic conjugates are typically prepared by coupling the polypeptide to a carrier protein (e.g., as a fusion protein) or, alternatively, they are recombinantly expressed in an immunization vector.

[0079] Monoclonal antibodies are prepared from cells secreting the desired antibody. These antibodies are screened for binding to normal or modified peptides, or screened for agonistic or antagonistic activity. Specific monoclonal and polyclonal antibodies will usually bind with a KD of at least about 50 mM, e.g., at least about 1 mM, e.g., at least about 0.1 mM or better. In some instances, it is desirable to prepare monoclonal antibodies from various mammalian hosts, such as rodents, lagomorphs, primates, humans, etc. Description of techniques for preparing such monoclonal antibodies are found in Kohler and Milstein 1975 Nature 256:495- 497. Summarized briefly, this method proceeds by injecting an animal with an immunogen, e.g., an immunogenic peptide either alone or optionally linked to a carrier protein. The animal is then sacrificed and cells taken from its spleen, which are fused with myeloma cells. The result is a hybrid cell or “hybridoma” that is capable of reproducing in vitro. The population of hybridomas is then screened to isolate individual clones, each of which secrete a single antibody species to the immunogen. In this manner, the individual antibody species obtained are the products of immortalized and cloned single B cells from the immune animal generated in response to a specific site recognized on the immunogenic substance.

[0080] Alternative methods of immortalization include transformation with Epstein Barr Virus, oncogenes, or retroviruses, or other methods known in the art. Colonies arising from single immortalized cells are screened for production of antibodies of the desired specificity and affinity for the antigen, and yield of the monoclonal antibodies produced by such cells is enhanced by various techniques, including injection into the peritoneal cavity of a vertebrate (preferably mammalian) host. The polypeptides and antibodies of the present invention are used with or without modification, and include chimeric antibodies such as humanized murine antibodies. Other suitable techniques involve selection of libraries of recombinant antibodies in phage or similar vectors. See, Huse et al. 1989 Science 246: 1275-1281; and Ward et al. 1989 Nature 341 :544-546. [0081] Antibodies specific to the target polypeptide can also be obtained by phage display techniques known in the art.

[0082] The present invention additionally provides polynucleotides encoding the binding agent of this invention. In some embodiments, the polynucleotides encode the polypeptides of any one of SEQ ID NOS :3- 18.

[0083] Further provided herein is a vector comprising the polynucleotide of the invention. Vectors include, but are not limited to, plasmid vectors, phage vectors, virus vectors, or cosmid vectors.

[0084] In some embodiments, the present invention provides a host cell comprising the polynucleotide and/or vector of this invention. The host cell can be a eukaryotic or prokaryotic cell and may be used for expressing the binding agent or other purposes.

[0085] A further aspect of the invention relates to a composition comprising the binding agent of the invention and a carrier. In some embodiments, the composition is a pharmaceutical composition and the carrier is a pharmaceutically acceptable carrier.

[0086] In some embodiments, the pharmaceutical composition may further comprise an additional therapeutic agent, e.g., a chemotherapeutic agent. Agents useful for treating cancer include, without limitation: 1) vinca alkaloids (e.g., vinblastine, vincristine); 2) epipodophyllotoxins (e.g., etoposide and teniposide); 3) antibiotics (e.g., dactinomycin (actinomycin D), daunorubicin (daunomycin; rubidomycin), doxorubicin, bleomycin, plicamycin (mithramycin), and mitomycin (mitomycin C)); 4) enzymes (e.g., L-asparaginase); 5) biological response modifiers (e.g., interferon-alfa); 6) platinum coordinating complexes (e.g., cisplatin and carboplatin); 7) anthracenediones (e.g., mitoxantrone); 8) substituted ureas (e.g., hydroxyurea); 9) methylhydrazine derivatives (e.g., procarbazine (N-methylhydrazine; MIH)); 10) adrenocortical suppressants (e.g., mitotane (o,p'-DDD) and aminoglutethimide); 11) adrenocorticosteroids (e.g., prednisone); 12) progestins (e.g., hydroxyprogesterone caproate, medroxyprogesterone acetate, and megestrol acetate); 13) estrogens (e.g., diethylstilbestrol and ethinyl estradiol); 14) antiestrogens (e.g., tamoxifen); 15) androgens (e.g., testosterone propionate and fluoxymesterone); 16) antiandrogens (e.g., flutamide): and 17) gonadotropin-releasing hormone analogs (e.g., leuprolide). In another embodiment, the agents of the invention are administered in conjunction with anti-angiogenesis agents, such as antibodies to VEGF (e.g., bevacizumab (AVASTIN), ranibizumab (LUCENTIS)) and other promoters of angiogenesis (e.g., bFGF, angiopoietin-1), antibodies to alpha-v/beta-3 vascular integrin (e.g., VITAXIN), angiostatin, endostatin, dalteparin, ABT-510, CNGRC peptide TNF alpha conjugate, cyclophosphamide, combretastatin A4 phosphate, dimethylxanthenone acetic acid, docetaxel, lenalidomide, enzastaurin, paclitaxel, paclitaxel albumin-stabilized nanoparticle formulation (Abraxane), soy isoflavone (Genistein), tamoxifen citrate, thalidomide, ADH-1 (EXHERIN), AG-013736, AMG-706, AZD2171, sorafenib tosylate, BMS-582664, CHIR-265, pazopanib, PI-88, vatalanib, everolimus, suramin, sunitinib malate, XL184, ZD6474, ATN-161, cilenigtide, and celecoxib, or any combination thereof. In other embodiments, the agents of the invention are administered in conjunction with one or more therapeutic antibodies, e.g., anti-cancer antibodies or antibodies to immune checkpoints. In other embodiments, the agents of the invention are administered in conjunction with one or more immune checkpoint inhibitors. The immune checkpoint inhibitor may be any molecule that inhibits an immune checkpoint. Immune checkpoints are well known in the art and include, without limitation, PD-1, PD-L1, PD-L2, CTLA4, B7-H3, B7-H4, BTLA, IDO, KIR, LAG3, A2AR, TIM-3, and VISTA. In some embodiments, the inhibitor is an antibody against the immune checkpoint protein. In certain embodiments, the immune checkpoint inhibitor is an inhibitor of PD-1 or PD-L1, e.g., an antibody that specifically binds PD-1 or PD-L1. In some embodiments, the immune checkpoint inhibitor is nivolumab, pembrolizumab, ipilimumab, durvalumab, or atezolizumab. In some embodiments, the binding agent may be directly or indirectly linked with an additional therapeutic agent to form an antibody drug conjugate.

[0087] Chemotherapeutic agents approved for treatment of neuroblastoma include granulocyte-macrophage colony-stimulating factor, interleukin-2, and 13-cis-retinoic acid. [0088] An additional aspect of the invention relates to a kit comprising the binding agent of the invention or cells for producing the binding agent of the invention. In some embodiments, the kit can include multiple binding agents and/or compositions containing such agents. In some embodiments, each of multiple binding agents provided in such a kit can specifically bind to a different antigen and/or engage a different myeloid-derived cell. In some embodiments, the kit can further include an additional active agent, e.g., a chemotherapeutic agent as would be known to one of skill in the art. In some embodiments, the kit can further include additional reagents, buffers, containers, etc.

Methods using binding agents

[0089] One aspect of the invention relates to a method of killing a cancer cell GD2 or a cancer cell expressing GD2 and CD47, comprising contacting the cell with an effective amount of the binding agent of the invention. [0090] An additional aspect of the invention relates to a method of targeting a macrophage to a cancer cell expressing GD2 or a cancer cell expressing GD2 and CD47, comprising contacting the cancer cell and the macrophage with an effective amount of the binding agent of the invention.

[0091] Another aspect of the invention relates to a method inducing macrophage-dependent antibody-dependent cellular cytotoxicity (ADCC) and/or antibody-dependent cellular phagocytosis (ADCP) against a cancer expressing GD2 or a cancer expressing GD2 and CD47 in a subject in need thereof, comprising administering a therapeutically effective amount of the binding agent or the pharmaceutical composition of the invention to the subject, thereby inducing macrophage-dependent ADCC and/or ADCP.

[0092] A further aspect of the invention relates to a method of treating a cancer expressing GD2 or a cancer expressing GD2 and CD47 in a subject in need thereof, comprising administering a therapeutically effective amount of the binding agent or the pharmaceutical composition of the invention to the subject, wherein the administering induces ADCC and/or ADCP of the cancer, thereby treating the cancer.

[0093] Another aspect of the invention relates to a method of treating a cancer in a subject in need thereof, comprising the steps of: a) selecting a subject having cancer cells that are enriched for GD2 or enriched for GD2 and CD47 and enriched for myeloid-derived cells (e.g., macrophages); and b) administering a therapeutically effective amount of the binding agent or the pharmaceutical composition of the invention to the subject, thereby treating the cancer.

[0094] The term “enriched”, as used herein, refers to a level of antigen on a cancer cell or level of myeloid-derived cells in a tumor that is greater than the level found in the cancer cell or tumor at an earlier point in time or greater than the average level found in similar cancer cells or tumors at a similar stage in the general population.

[0095] The selection step may be carried out by any method known to measure antigens and cells. In some embodiments, step a) comprises obtaining a sample of the cancer from the subject and measuring the level of antigen and myeloid-derived cells in the sample. The level of antigen may be measured by, e.g., an immunoassay, protein analysis, RNA analysis, or immunohistochemistry. The level of myeloid-derived cells may be measured by, e.g., an immunoassay, protein analysis, RNA analysis, or flow cytometry.

[0096] A myeloid-derived cell that accumulates in tumors is a cell type that is enriched in tumors. In some embodiments, the level of the myeloid-derived cell in the tumor increases by 2-fold, 5-fold, 10-fold or more relative to the level before the transition. In some embodiments, the myeloid-derived cell is a macrophage, dendritic cell, or a granulocyte, such as a neutrophil, basophil, eosinophil, or mast cell. In some embodiments, the myeloid-derived cell is a macrophage.

[0097] In some embodiments, the cancer is a mesenchymal tumor. In some embodiments, the cancer is neuroblastoma, glioblastoma, retinoblastoma, breast cancer (e.g., triple negative breast cancer), bladder cancer, melanoma, lung cancer, osteosarcoma, or Ewing sarcoma.

[0098] In some embodiments, the methods may further comprise the step of isolating myeloid-derived cells from the subject, contacting the myeloid-derived cells with the binding agent or pharmaceutical composition, and administering the contacted myeloid-derived cells to the subject.

[0099] In some embodiments, more than one binding agent may be delivered to a subject. For example, if a cancer sample shows expression of more than one targetable antigen or more than one type of myeloid-derived cell is enriched in the cancer, agents targeting each of the antigens and/or myeloid-derived cells may be administered. In some embodiments, the binding agent may be multispecific (e.g., trispecific) in order to engage multiple targetable antigens and/or more than one type of myeloid-derived cell.

[0100] The methods of the invention may further comprise administering to the subject an additional cancer therapeutic agent or treatment (e.g., surgery, radiation). Cancer therapeutic agents include, without limitation, 1) vinca alkaloids (e.g., vinblastine, vincristine); 2) epipodophyllotoxins (e.g., etoposide and teniposide); 3) antibiotics (e.g., dactinomycin (actinomycin D), daunorubicin (daunomycin; rubidomycin), doxorubicin, bleomycin, plicamycin (mithramycin), and mitomycin (mitomycin C)); 4) enzymes (e.g., L-asparaginase); 5) biological response modifiers (e.g., interferon-alfa); 6) platinum coordinating complexes (e.g., cisplatin and carboplatin); 7) anthracenediones (e.g., mitoxantrone); 8) substituted ureas (e.g., hydroxyurea); 9) methylhydrazine derivatives (e.g., procarbazine (N-methylhydrazine; MH4)); 10) adrenocortical suppressants (e.g., mitotane (o,p'-DDD) and aminoglutethimide); 11) adrenocorticosteroids (e.g., prednisone); 12) progestins (e.g., hydroxyprogesterone caproate, medroxyprogesterone acetate, and megestrol acetate); 13) estrogens (e.g., diethylstilbestrol and ethinyl estradiol); 14) antiestrogens (e.g., tamoxifen); 15) androgens (e.g., testosterone propionate and fluoxymesterone); 16) antiandrogens (e.g., flutamide): and 17) gonadotropin-releasing hormone analogs (e.g., leuprolide). Other cancer therapeutic agents include, without limitation, anti-angiogenesis agents, such as antibodies to VEGF (e.g., bevacizumab (AVASTIN), ranibizumab (LUCENTIS)) and other promoters of angiogenesis (e.g., bFGF, angiopoietin-1), angiostatin, endostatin, dalteparin, ABT-510, CNGRC peptide TNF alpha conjugate, cyclophosphamide, combretastatin A4 phosphate, dimethylxanthenone acetic acid, docetaxel, lenalidomide, enzastaurin, paclitaxel, paclitaxel albumin-stabilized nanoparticle formulation (Abraxane), soy isoflavone (Genistein), tamoxifen citrate, thalidomide, ADH-1 (EXHERIN), AG-013736, AMG-706, AZD2171, sorafenib tosylate, BMS-582664, CHIR-265, pazopanib, PI-88, vatalanib, everolimus, suramin, sunitinib malate, XL 184, ZD6474, ATN-161, cilenigtide, and celecoxib.

[0101] In some embodiments, the methods further comprise administering to the subject an immune checkpoint inhibitor. Immune checkpoints are well known in the art and include, without limitation, PD-1, PD-L1, PD-L2, CTLA4, B7-H3, B7-H4, BTLA, IDO, KIR, LAG3, A2AR, TIM-3, and VISTA. In some embodiments, the inhibitor is an antibody against the immune checkpoint protein. In certain embodiments, the immune checkpoint inhibitor is an inhibitor of PD-1, PD-L1, or CTLA-4 that are enriched in mesenchymal tumors, e.g., an antibody that specifically binds PD-1, PD-L1, or CTLA-4. In some embodiments, the immune checkpoint inhibitor is nivolumab, pembrolizumab, ipilimumab, durvalumab, or atezolizumab. [0102] Cancer therapeutic agents approved for treatment of neuroblastoma include granulocyte-macrophage colony-stimulating factor, interleukin-2, and 13-cis-retinoic acid.

[0103] In certain embodiments, the binding agents used in the methods of the present invention are administered directly to a subject. In some embodiments, the binding agents will be suspended in a pharmaceutically-acceptable carrier e.g., physiological saline) and administered orally or by intravenous infusion, or administered subcutaneously, intramuscularly, intrathecally, intraperitoneally, intrarectally, intravaginally, intranasally, intragastrically, intratracheally, or intrapulmonarily. In another embodiment, the intratracheal or intrapulmonary delivery can be accomplished using a standard nebulizer, jet nebulizer, wire mesh nebulizer, dry powder inhaler, or metered dose inhaler. The agents can be delivered directly to the site of the disease or disorder, such as lungs, kidney, or intestines, e.g., injected in situ into or near a tumor. The dosage required depends on the choice of the route of administration; the nature of the formulation; the nature of the patient’s illness; the subject’s size, weight, surface area, age, and sex; other drugs being administered; and the judgment of the attending physician. Suitable dosages for each agent are in the range of 0.01-100 pg/kg. Wide variations in the needed dosage are to be expected in view of the variety of agents available and the differing efficiencies of various routes of administration. For example, oral administration would be expected to require higher dosages than administration by i.v. injection. Variations in these dosage levels can be adjusted using standard empirical routines for optimization as is well understood in the art. Administrations can be single or multiple e.g., 2-, 3-, 4-, 6-, 8-, 10-; 20-, 50-, 100-, 150-, or more fold). Encapsulation of the compound in a suitable delivery vehicle (e.g., polymeric microparticles or nanoparticles or implantable devices) may increase the efficiency of delivery, particularly for oral delivery.

[0104] By “pharmaceutically acceptable” it is meant a material that is not biologically or otherwise undesirable, i.e., the material can be administered to a subject without causing any undesirable biological effects such as toxicity.

[0105] The formulations of the invention can optionally comprise medicinal agents, pharmaceutical agents, carriers, adjuvants, dispersing agents, diluents, and the like.

[0106] The binding agents of the invention can be formulated for administration in a pharmaceutical carrier in accordance with known techniques. See, e.g., Remington, The Science and Practice of Pharmacy (21 ^st Ed. 2006). In the manufacture of a pharmaceutical formulation according to the invention, the agent is typically admixed with, inter alia, an acceptable carrier. The carrier can be a solid or a liquid, or both, and may be formulated with the agent as a unit-dose formulation, for example, a capsule or vial, which can contain from 0.01 or 0.5% to 95% or 99% by weight of the agent. One or more agents can be incorporated in the formulations of the invention, which can be prepared by any of the well-known techniques of pharmacy.

[0107] The formulations of the invention include those suitable for oral, rectal, topical, buccal (e.g., sub-lingual), vaginal, parenteral (e.g., subcutaneous, intramuscular including skeletal muscle, cardiac muscle, diaphragm muscle and smooth muscle, intradermal, intravenous, intraperitoneal), topical i.e., both skin and mucosal surfaces, including airway surfaces), intranasal, transdermal, intraarticular, intrathecal, and inhalation administration, administration to the liver by intraportal delivery, as well as direct organ injection (e.g., into the liver, into the brain for delivery to the central nervous system, or into the pancreas) or injection into a body cavity. The most suitable route in any given case will depend on the nature and severity of the condition being treated and on the nature of the particular agent which is being used.

[0108] For injection, the carrier will typically be a liquid, such as sterile pyrogen-free water, pyrogen-free phosphate-buffered saline solution, bacteriostatic water, or Cremophor EL[R] (BASF, Parsippany, N.J.). For other methods of administration, the carrier can be either solid or liquid.

[0109] For oral administration, the agent can be administered in solid dosage forms, such as capsules, tablets, and powders, or in liquid dosage forms, such as elixirs, syrups, and suspensions. Agents can be encapsulated in gelatin capsules together with inactive ingredients and powdered carriers, such as glucose, lactose, sucrose, mannitol, starch, cellulose or cellulose derivatives, magnesium stearate, stearic acid, sodium saccharin, talcum, magnesium carbonate and the like. Examples of additional inactive ingredients that can be added to provide desirable color, taste, stability, buffering capacity, dispersion or other known desirable features are red iron oxide, silica gel, sodium lauryl sulfate, titanium dioxide, edible white ink, and the like. Similar diluents can be used to make compressed tablets. Both tablets and capsules can be manufactured as sustained release products to provide for continuous release of medication over a period of hours. Compressed tablets can be sugar coated or film coated to mask any unpleasant taste and protect the tablet from the atmosphere, or enteric- coated for selective disintegration in the gastrointestinal tract. Liquid dosage forms for oral administration can contain coloring and flavoring to increase patient acceptance.

[0110] Formulations suitable for buccal (sub-lingual) administration include lozenges comprising the agent in a flavored base, usually sucrose and acacia or tragacanth; and pastilles comprising the agent in an inert base such as gelatin and glycerin or sucrose and acacia.

[OHl] Formulations of the present invention suitable for parenteral administration comprise sterile aqueous and non-aqueous injection solutions of the agent, which preparations are preferably isotonic with the blood of the intended recipient. These preparations can contain antioxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient. Aqueous and non-aqueous sterile suspensions can include suspending agents and thickening agents. The formulations can be presented in unit/dose or multi-dose containers, for example sealed ampoules and vials, and can be stored in a freeze- dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, saline or water-for-inj ection immediately prior to use.

[0112] Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules and tablets of the kind previously described. For example, in one aspect of the present invention, there is provided an injectable, stable, sterile composition comprising an agent of the invention, in a unit dosage form in a sealed container. The agent is provided in the form of a lyophilizate which is capable of being reconstituted with a suitable pharmaceutically acceptable carrier to form a liquid composition suitable for injection thereof into a subject. The unit dosage form typically comprises from about 1 mg to about 10 grams of the agent. When the agent is substantially water-insoluble, a sufficient amount of emulsifying agent which is pharmaceutically acceptable can be employed in sufficient quantity to emulsify the agent in an aqueous carrier. One such useful emulsifying agent is phosphatidyl choline. [0113] Formulations suitable for rectal administration are preferably presented as unit dose suppositories. These can be prepared by admixing the agent with one or more conventional solid carriers, for example, cocoa butter, and then shaping the resulting mixture.

[0114] Formulations suitable for topical application to the skin preferably take the form of an ointment, cream, lotion, paste, gel, spray, aerosol, or oil. Carriers which can be used include petroleum jelly, lanoline, polyethylene glycols, alcohols, transdermal enhancers, and combinations of two or more thereof.

[0115] Formulations suitable for transdermal administration can be presented as discrete patches adapted to remain in intimate contact with the epidermis of the recipient for a prolonged period of time. Formulations suitable for transdermal administration can also be delivered by iontophoresis (see, for example, Tyle, Pharm. Res. 3:318 (1986)) and typically take the form of an optionally buffered aqueous solution of the compounds. Suitable formulations comprise citrate or bis/tris buffer (pH 6) or ethanol/water and contain from 0.1 to 0.2M of the compound. [0116] The agent can alternatively be formulated for nasal administration or otherwise administered to the lungs of a subject by any suitable means, e.g., administered by an aerosol suspension of respirable particles comprising the agent, which the subject inhales. The respirable particles can be liquid or solid. The term “aerosol” includes any gas-borne suspended phase, which is capable of being inhaled into the bronchioles or nasal passages. Specifically, aerosol includes a gas-borne suspension of droplets, as can be produced in a metered dose inhaler or nebulizer, or in a mist sprayer. Aerosol also includes a dry powder composition suspended in air or other carrier gas, which can be delivered by insufflation from an inhaler device, for example. See Ganderton & Jones, Drug Delivery to the Respiratory Tract, Ellis Horwood (1987); Gonda (1990) Critical Reviews in Therapeutic Drug Carrier Systems 6:273-313; and Raeburn et al., J. Pharmacol. Toxicol. Meth. 27:143 (1992). Aerosols of liquid particles comprising the agent can be produced by any suitable means, such as with a pressure-driven aerosol nebulizer or an ultrasonic nebulizer, as is known to those of skill in the art. See, e.g., U.S. Patent No. 4,501,729. Aerosols of solid particles comprising the agent can likewise be produced with any solid particulate medicament aerosol generator, by techniques known in the pharmaceutical art.

[0117] Alternatively, one can administer the agent in a local rather than systemic manner, for example, in a depot or sustained-release formulation.

[0118] Further, the present invention provides liposomal formulations of the agents disclosed herein and salts thereof. The technology for forming liposomal suspensions is well known in the art. When the compound or salt thereof is an aqueous-soluble salt, using conventional liposome technology, the same can be incorporated into lipid vesicles. In such an instance, due to the water solubility of the agent, the agent will be substantially entrained within the hydrophilic center or core of the liposomes. The lipid layer employed can be of any conventional composition and can either contain cholesterol or can be cholesterol-free. When the compound or salt of interest is water-insoluble, again employing conventional liposome formation technology, the salt can be substantially entrained within the hydrophobic lipid bilayer which forms the structure of the liposome. In either instance, the liposomes which are produced can be reduced in size, as through the use of standard sonication and homogenization techniques.

[0119] The liposomal formulations containing the agent can be lyophilized to produce a lyophilizate which can be reconstituted with a pharmaceutically acceptable carrier, such as water, to regenerate a liposomal suspension.

[0120] In the case of water-insoluble agents, a pharmaceutical composition can be prepared containing the water-insoluble agent, such as for example, in an aqueous base emulsion. In such an instance, the composition will contain a sufficient amount of pharmaceutically acceptable emulsifying agent to emulsify the desired amount of the agent. Particularly useful emulsifying agents include phosphatidyl cholines and lecithin.

[0121] In particular embodiments, the agent is administered to the subject in a therapeutically effective amount, as that term is defined above. Dosages of pharmaceutically active agents can be determined by methods known in the art, see, e.g., Remington's Pharmaceutical Sciences (Maack Publishing Co., Easton, Pa). The therapeutically effective dosage of any specific agent will vary somewhat from agent to agent, and patient to patient, and will depend upon the condition of the patient and the route of delivery. As a general proposition, a dosage from about 0.1 to about 50 mg/kg will have therapeutic efficacy, with all weights being calculated based upon the weight of the agent. Toxicity concerns at the higher level can restrict intravenous dosages to a lower level such as up to about 10 mg/kg, with all weights being calculated based upon the weight of the agent. A dosage from about 10 mg/kg to about 50 mg/kg can be employed for oral administration. Typically, a dosage from about 0.5 mg/kg to 5 mg/kg can be employed for intramuscular injection. Particular dosages are about 1 pmol/kg to 50 pmol/kg, and more particularly to about 22 pmol/kg and to 33 pmol/kg of the agent for intravenous or oral administration, respectively. [0122] In particular embodiments of the invention, more than one administration e.g., two, three, four, or more administrations) can be employed over a variety of time intervals (e.g., hourly, daily, weekly, monthly, etc.) to achieve therapeutic effects.

[0123] The present invention finds use in veterinary and medical applications. Suitable subjects include both avians and mammals, with mammals being preferred. The term “mammal” as used herein includes, but is not limited to, humans, primates, bovines, ovines, caprines, equines, felines, canines, lagomorphs, etc. Human subjects include neonates, infants, juveniles, and adults. The subject may be one in need of the methods of the invention, e.g., a subject that has or is suspected of having cancer. The subject may be a laboratory animal, e.g., an animal model of a disease.

[0124] The present invention is more particularly described in the following examples that are intended as illustrative only since numerous modifications and variations therein will be apparent to those skilled in the art.

Example 1

[0125] Antibody therapeutics that are engineered to engage NK cells for tumor cell killing are limited in utility for tumors with an immune-cold phenotype for which NK cells and other anti-tumor immune cells are excluded. As shown in FIG. 1, neuroblastoma tumors are enriched for macrophage markers, while the expression of markers that identify NK cells, neutrophils, and dendritic cells are much lower. As such, we propose that monoclonal antibodies designed to selectively engage macrophages would be ideally suited for neuroblastoma therapy.

[0126] To test this hypothesis, we engineered multiple forms of an antibody targeting a cell surface marker found on neuroblastoma cells, the disialoganglioside GD2. GD2 is a particularly good target because it is highly enriched on the surface of neuroblastoma cells, while its expression is otherwise highly restricted and only found on limited normal cells of neuroectodermal origin. An antibody targeting GD2, dinutuximab/Unituxin has been developed and FDA-approved for use in pediatric neuroblastoma. We first confirmed that switching the isotype of dinutuximab would not alter its binding to neuroblastoma cells. FIG. 2 confirms that both the hIgG4 and hlgGl forms of anti-GD2 show equivalent affinity for GD2 on SK-NA-S neuroblastoma cells by flow cytometry.

[0127] Since neuroblastoma tumors are enriched for macrophages but depleted of NK cells, our goal was to create an antibody capable of inducing macrophage-mediated killing that would not engage other types of immune effector cells, as this selective engagement should promote antibody accumulation within the tumor and avoid binding to circulating immune cells. We first confirmed that h!gG4 anti-GD2 is able to induce macrophage-mediated killing of SK-N- AS and SHEP neuroblastoma cells (FIGS. 4, 6, and 7). As expected, only the hlgGl but not the hIgG4 form of anti-GD2 is able to engage NK cells to mediate killing of SHEP neuroblastoma cells (FIG. 5). Thus, hIgG4 anti-GD2 is capable of binding to and inducing in vitro macrophage mediated killing of human neuroblastoma cells.

[0128] In addition to neuroblastoma, GD2 expression has been found on breast cancer, melanoma, and small cell lung cancer. In fact, GD2 has been identified as a marker of breast cancer stem cells and a driver of tumorigenesis (PMID 22585577). Compared to hlgGl anti- GD2, hIgG4 anti-GD2 produced a higher level of macrophage-mediated killing of GD2- expressing HCC1395 and Hs578T human triple negative breast cancer cells in vitro (FIG. 8). [0129] The foregoing is illustrative of the present invention, and is not to be construed as limiting thereof. The invention is defined by the following claims, with equivalents of the claims to be included therein.

References:

Carter, P. J. and G.A. Lazar, Next generation antibody drugs: pursuit of the 'high-hanging fruit'. Nat Rev Drug Discov, 17(3): p. 197-223, 2018.

Chiavenna, S.M., J.P. Jaworski, and A. Vendrell, State of the art in anti-cancer mAbs. Journal of biomedical science, 24(1): p. 15-15, 2017.

Gasser, M. and A.M. Waaga-Gasser, Therapeutic Antibodies in Cancer Therapy. Adv Exp Med Biol, 917: p. 95-120, 2016.

Lazar, G.A., W. Dang, S. Karki, O. Vafa, J.S. Peng, L. Hyun, C. Chan, H.S. Chung, A. Eivazi, S.C. Yoder, J. Vielmetter, D.F. Carmichael, R.J. Hayes, and B.I. Dahiyat, Engineered antibody Fc variants with enhanced effector function. Proceedings of the National Academy of Sciences of the United States of America, 103(11): p. 4005-4010, 2006.

Li, B., L. Xu, C. Pi, Y. Yin, K. Xie, F. Tao, R. Li, H. Gu, and J. Fang, CD89-mediated recruitment of macrophages via a bispecific antibody enhances anti-tumor efficacy. Oncoimmunology, 7(1): p. e!380142-el380142, 2017.

Rader, C., Bispecific antibodies in cancer immunotherapy. Curr Opin Biotechnol, 65: p. 9-16, 2020. Richards, J.O., S. Karki, G.A. Lazar, H. Chen, W. Dang, and J.R. Desjarlais, Optimization of antibody binding to FcgammaRIIa enhances macrophage phagocytosis of tumor cells. Mol Cancer Ther, 7(8): p. 2517-27, 2008.

Saxena, A. and D. Wu, Advances in Therapeutic Fc Engineering - Modulation of IgG- Associated Effector Functions and Serum Half-life. Frontiers in immunology, 7: p. 580-580, 2016.

Strop, P. et al., Location matters: Site of conjugation modulates stability and pharmacokinetics of antibody drug conjugates. Chem. Biol. 20: 161-167, 2013.

Wilkinson, I. et al., Fc-engineered antibodies with immune effector functions completely abolished. PLos ONE 16(12): e0260954, 2021.

Sequences:

ABT-200.0 dinutuximab hlgGl

Heavy chain (evi-5 dinu.VH-gm3.HC) (SEQ ID NO:1)

EVQLLQSGPELEKPGASVMISCKASGSSFTGYNMNWVRQNIGKSLEWIGAIDPYYGG TSYNQKFKGRA TLTVDKSSSTAYMHLKSLTSEDSAVYYCVSGMEYWGQGTSVTVSSASTKGPSVFPLAPSS KSTSGGTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICN VNHKPSNT KVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHED PEVKFNWY VDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA KGQPRE PQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSKLTVDK

SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Light chain (evi-5 dinu.VL-hk.LC) (SEQ ID NO:2)

EIVMTQSPATLSVSPGERATLSCRSSQSLVHRNGNTYLHWYLQKPGQSPKLLIHKVS NRFSGVPDRFSG SGSGTDFTLKISRVEAEDLGVYFCSQSTHVPPLTFGAGTKLELKRTVAAPSVFIFPPSDE QLKSGTASW CLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA CEVTHQG LSSPVTKSFNRGEC

ABT-201.0 dinutuximab hIgG4 S228P

Heavy chain (evi-5 dinu.VH-h4.HC.S228P) (SEQ ID NO:3)

EVQLLQSGPELEKPGASVMISCKASGSSFTGYNMNWVRQNIGKSLEWIGAIDPYYGG TSYNQKFKGRA TLTVDKSSSTAYMHLKSLTSEDSAVYYCVSGMEYWGQGTSVTVSSASTKGPSVFPLAPCS RSTSESTAA LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTKTYTCNV DHKPSNTK VDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSQEDPEVQ FNWYVDGV EVHNAKTKPREEQFNSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQP REPQVYT LPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPE Light chain (evi-5 dinu.VL-hk.LC) (SEQ ID NO:4)

EIVMTQSPATLSVSPGERATLSCRSSQSLVHRNGNTYLHWYLQKPGQSPKLLIHKVS NRFSGVPDRFSG SGSGTDFTLKISRVEAEDLGVYFCSQSTHVPPLTFGAGTKLELKRTVAAPSVFIFPPSDE QLKSGTASVV CLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA CEVTHQG

LSSPVTKSFNRGEC

ABT-201.1 dinutuximab hIgG4 S228P LC-SIRPa (SIRPa-dinutuxi IgG4)

Heavy chain (PRO 45212_evi-5 dinu.VH-h4.HC.S228P) (SEQ ID NO:5)

EVQLLQSGPELEKPGASVMISCKASGSSFTGYNMNWVRQNIGKSLEWIGAIDPYYGG TSYNQKFKGRA

TLTVDKSSSTAYMHLKSLTSEDSAVYYCVSGMEYWGQGTSVTVSSASTKGPSVFPLA PCSRSTSESTAA

LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTKTYT CNVDHKPSNTK

VDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSQEDP EVQFNWYVDGV EVHNAKTKPREEQFNSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQP REPQVYT LPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRL TVDKSRWQ

EGNVFSCSVMHEALHNHYTQKSLSLSLGK

Light chain (evi-5 SIRPa-(G4S)3-dinutuxi.VL-hk(LELK).LC) (SEQ ID NO:6)

EEELQVIQPDKSVSVAAGESAILHCTVTSLIPVGPIQWFRGAGPARELIYNQKEGHF PRVTTVSESTKRE

NMDFSISISNITPADAGTYYCVKFRKGSPDTEFKSGAGTELSVGGGGSGGGGSGGGG SEIVMTQSPATL SVSPGERATLSCRSSQSLVHRNGNTYLHWYLQKPGQSPKLLIHKVSNRFSGVPDRFSGSG SGTDFTLKIS RVEAEDLGVYFCSQSTHVPPLTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASWCLL NNFYPREA

KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL SSPVTKSFNR GEC

ABT-201.2 - dinutuximab hIgG4 S228P LC-SIRPa-D (formerly SIRPalphaD-dinutuxi

IgG4)

Heavy chain (PRO 45212_evi-5 dinu.VH-h4.HC.S228P) (SEQ ID NO:7)

EVQLLQSGPELEKPGASVMISCKASGSSFTGYNMNWVRQNIGKSLEWIGAIDPYYGG TSYNQKFKGRA

TLTVDKSSSTAYMHLKSLTSEDSAVYYCVSGMEYWGQGTSVTVSSASTKGPSVFPLA PCSRSTSESTAA

LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTKTYT CNVDHKPSNTK

VDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSQEDP EVQFNWYVDGV EVHNAKTKPREEQFNSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQP REPQVYT LPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRL TVDKSRWQ

EGNVFSCSVMHEALHNHYTQKSLSLSLGK

Light chain (evi-5 SIRPa.mod-(G4S)3-dinutuxi.VL-hk(LELK).LC) (SEQ ID NO:8) DDDDLQVIQPDKSVSVAAGESAILHCTVTSLIPVGPIQWFRGAGPARELIYNQKEGHFPR VTTVSESTKR

ENMDFSISISNITPADAGTYYCVKFRKGSPDTEFKSGAGTELSVKAKPSGGGGSGGG GSGGGGSEIVMT

QSPATLSVSPGERATLSCRSSQSLVHRNGNTYLHWYLQKPGQSPKLLIHKVSNRFSG VPDRFSGSGSGT

DFTLKISRVEAEDLGVYFCSQSTHVPPLTFGAGTKLELKRTVAAPSVFIFPPSDEQL KSGTASVVCLLNN

FYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVT

KSFNRGEC

ABT-201.3 dinutuximab hIgG4 S228P LC-SIRPa-TGl (SIRPaTGl-dinutuxi IgG4)

Heavy chain (PRO 45212_evi-5 dinu.VH-h4.HC.S228P) (SEQ ID NO:9)

EVQLLQSGPELEKPGASVMISCKASGSSFTGYNMNWVRQNIGKSLEWIGAIDPYYGG TSYNQKFKGRA

TLTVDKSSSTAYMHLKSLTSEDSAVYYCVSGMEYWGQGTSVTVSSASTKGPSVFPLA PCSRSTSESTAA

LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTKTYT CNVDHKPSNTK

VDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSQEDP EVQFNWYVDGV

EVHNAKTKPREEQFNSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAK GQPREPQVYT

LPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SRLTVDKSRWQ

EGNVFSCSVMHEALHNHYTQKSLSLSLGK

Light chain (evi-5 SIRPa.TGl-(G4S)3-dinutuxi.VL-hk(LELK).LC) (SEQ ID NO:10)

EEELQVIQPDKSVSVAAGLLQGESAILHCTVTSLIPVGPIQWFRGAGPARELIYNQK EGHFPRVTTVSES

TKRENMDFSISISNITPADAGTYYCVKFRKGSPDTEFKSGAGTELSVGGGGSGGGGS GGGGSEIVMTQS

PATLSVSPGERATLSCRSSQSLVHRNGNTYLHWYLQKPGQSPKLLIHKVSNRFSGVP DRFSGSGSGTDF

TLKISRVEAEDLGVYFCSQSTHVPPLTFGAGTKLELKRTVAAPSVFIFPPSDEQLKS GTASWCLLNNFY

PREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVT HQGLSSPVTKS

FNRGEC

ABT-201.4 dinutuximab hIgG4 S228P LC-SIRPa-TG2 (SIRPaTG2-dinutuxi IgG4)

Heavy chain (PRO 45212_evi-5 dinu.VH-h4.HC.S228P) (SEQ ID NO:11)

EVQLLQSGPELEKPGASVMISCKASGSSFTGYNMNWVRQNIGKSLEWIGAIDPYYGG TSYNQKFKGRA

TLTVDKSSSTAYMHLKSLTSEDSAVYYCVSGMEYWGQGTSVTVSSASTKGPSVFPLA PCSRSTSESTAA

LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTKTYT CNVDHKPSNTK

VDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSQEDP EVQFNWYVDGV

EVHNAKTKPREEQFNSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAK GQPREPQVYT

LPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SRLTVDKSRWQ

EGNVFSCSVMHEALHNHYTQKSLSLSLGK

Light chain (evi-5 SIRPa.TG2-(G4S)3-dinutuxi.VL-hk(LELK).LC) (SEQ ID NO: 12)

EEELQVIQPDKSVSVAAGESAILHCTVTSLIPVGPIQWFRGAGPARELIYNQKEGHF PRVTTVSESTKRE

NMDFSISISNITLLQGPADAGTYYCVKFRKGSPDTEFKSGAGTELSVGGGGSGGGGS GGGGSEIVMTQS PATLSVSPGERATLSCRSSQSLVHRNGNTYLHWYLQKPGQSPKLLIHKVSNRFSGVPDRF SGSGSGTDF TLKISRVEAEDLGVYFCSQSTHVPPLTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTA SWCLLNNFY PREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKS

FNRGEC

ABT-201.5 dinutuximab hIgG4 S228P LC-SIRPa-R69A (SIRPa-dinutuxi IgG4 R69A)

Heavy chain (PRO 45212_evi-5 dinu.VH-h4.HC.S228P) (SEQ ID NO:13)

EVQLLQSGPELEKPGASVMISCKASGSSFTGYNMNWVRQNIGKSLEWIGAIDPYYGG TSYNQKFKGRA

TLTVDKSSSTAYMHLKSLTSEDSAVYYCVSGMEYWGQGTSVTVSSASTKGPSVFPLA PCSRSTSESTAA

LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTKTYT CNVDHKPSNTK

VDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSQEDP EVQFNWYVDGV EVHNAKTKPREEQFNSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQP REPQVYT LPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRL TVDKSRWQ

EGNVFSCSVMHEALHNHYTQKSLSLSLGK

Light chain (evi-5 SIRPa.R69A-(G4S)3-dinutuxi.VL-hk(LELK).LC) (SEQ ID NO: 14)

EEELQVIQPDKSVSVAAGESAILHCTVTSLIPVGPIQWFRGAGPARELIYNQKEGHF PRVTTVSESTKAE

NMDFSISISNITPADAGTYYCVKFRKGSPDTEFKSGAGTELSVGGGGSGGGGSGGGG SEIVMTQSPATL SVSPGERATLSCRSSQSLVHRNGNTYLHWYLQKPGQSPKLLIHKVSNRFSGVPDRFSGSG SGTDFTLKIS RVEAEDLGVYFCSQSTHVPPLTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASWCLL NNFYPREA

KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL SSPVTKSFNR GEC

ABT-201.6 dinutuximab hIgG4 S228P LC-SIRPa-DlOOA (SIRPa-dinutuxi IgG4 D100A)

Heavy chain (PRO 45212_evi-5 dinu.VH-h4.HC.S228P) (SEQ ID NO:15)

EVQLLQSGPELEKPGASVMISCKASGSSFTGYNMNWVRQNIGKSLEWIGAIDPYYGG TSYNQKFKGRA

TLTVDKSSSTAYMHLKSLTSEDSAVYYCVSGMEYWGQGTSVTVSSASTKGPSVFPLA PCSRSTSESTAA

LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTKTYT CNVDHKPSNTK

VDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSQEDP EVQFNWYVDGV EVHNAKTKPREEQFNSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQP REPQVYT LPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRL TVDKSRWQ

EGNVFSCSVMHEALHNHYTQKSLSLSLGK

Light chain (evi-5 SIRPa.D100A-(G4S)3-dinutuxi.VL-hk(LELK).LC) (SEQ ID NO:16)

EEELQVIQPDKSVSVAAGESAILHCTVTSLIPVGPIQWFRGAGPARELIYNQKEGHF PRVTTVSESTKRE

NMDFSISISNITPADAGTYYCVKFRKGSPATEFKSGAGTELSVGGGGSGGGGSGGGG SEIVMTQSPATL SVSPGERATLSCRSSQSLVHRNGNTYLHWYLQKPGQSPKLLIHKVSNRFSGVPDRFSGSG SGTDFTLKIS RVEAEDLGVYFCSQSTHVPPLTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASWCLL NNFYPREA KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP VTKSFNR

GEC

SIRPalpha IgG4 S228P

Heavy chain (evi-5 h4.HC.S228P) (SEQ ID NO: 17)

ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL QSSGLYSLSSWT

VPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKP KDTLMISRTPEVT

CVWDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRWSVLTVLHQDWLNGKEY KCKVSN KGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTP PVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

Light chain (evi-5 SIRPa.mod-(G4S)3-hk.LC) (SEQ ID NO: 18)

EEELQVIQPDKSVSVAAGESAILHCTVTSLIPVGPIQWFRGAGPARELIYNQKEGHF PRVTTVSESTKRE

NMDFSISISNITPADAGTYYCVKFRKGSPDTEFKSGAGTELSVRAKPSGGGGSGGGG SGGGGSRTVAAP

SVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDS TYSLSSTLTLS

KADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

SIRPa wild-type fragment (SEQ ID NO: 19)

EEELQVIQPDKSVSVAAGESAILHCTVTSLIPVGPIQWFRGAGPARELIYNQKEGHF PRVTTVSESTKRE

NMDFSISISNITPADAGTYYCVKFRKGSPDTEFKSGAGTELSVRAKPS

SIRPa modified fragment (SEQ ID NO:20)

DDDDLQVIQPDKSVSVAAGESAILHCTVTSLIPVGPIQWFRGAGPARELIYNQKEGH FPRVTTVSESTKR

ENMDFSISISNITPADAGTYYCVKFRKGSPDTEFKSGAGTELSVRAKPS

SIRPa truncated fragment (SEQ ID NO:21)

EEELQVIQPDKSVSVAAGESAILHCTVTSLIPVGPIQWFRGAGPARELIYNQKEGHF PRVTTVSESTKRE

NMDFSISISNITPADAGTYYCVKFRKGSPDTEFKSGAGTELSV

SIRPa truncated TGI fragment (SEQ ID NO:22)

EEELQVIQPDKSVSVAAGLLQGESAILHCTVTSLIPVGPIQWFRGAGPARELIYNQK EGHFPRVTTVSES

TKRENMDFSISISNITPADAGTYYCVKFRKGSPDTEFKSGAGTELSV

SIRPa truncated TG2 fragment (SEQ ID NO:23)

EEELQVtQPDKSVSVAAGESAILHCTVTSLIPVGPIQWFRGAGPARELIYNQKEGHF PRVTTVSESTKRE

NMDFSISISNITLLQGPADAGTYYCVKFRKGSPDTEFKSGAGTELSV SIRPa truncated TG2del fragment (SEQ ID NO:24)

EEELQVIQPDKSVSVAAGESAILHCTVTSLIPVGPIQWFRGAGPARELIYNQKEGHF PRVTTVSESTKRE NMDFSISILLQGPADAGTYYCVKFRKGSPDTEFKSGAGTELSV

SIRPa truncated R69A fragment (SEQ ID NO: 25)

EEELQVIQPDKSVSVAAGESAILHCTVTSLIPVGPIQWFRGAGPARELIYNQKEGHF PRVTTVSESTKAE NMDFSISISNITPADAGTYYCVKFRKGSPDTEFKSGAGTELSV

SIRPa truncated DI 00 A fragment (SEQ ID NO: 26)

EEELQVIQPDKSVSVAAGESAILHCTVTSLIPVGPIQWFRGAGPARELIYNQKEGHF PRVTTVSESTKRE NMDFSISISNITPADAGTYYCVKFRKGSPATEFKSGAGTELSV

Transglutaminase conjugation site (SEQ ID NO:27)

LLQG

SIRPa deletion (SEQ ID NO:28)

SNITPADA

CrossMab KIH IgG4 variant 1 with normal etaracizumab Fab and crossed dinutuximab Fab (4 different chains)

Etaracizumab HC (hole) [S228P.T366S.T368A.Y407V] (SEQ ID NO:29)

QVQLVESGGGWQPGRSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVAKVSSGGGST YYLDTVQG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCARHLHGSFASWGQGTTVTVSSASTKGPSVF PLAPCSRST SESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTK TYTCNVDH KPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSQ EDPEVQFN WYVDGVEVHNAKTKPREEQFNSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTIS KAKGQP REPQVYTLPPSQEEMTKNQVSLSCLAKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLVSRLTV

DKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

Etaracizumab LC (normal) (SEQ ID NO: 30)

> PRO 34054_evi-5 ETAR VH-hk.LC

EIVLTQSPATLSLSPGERATLSCQASQSISNFLHWYQQRPGQAPRLLIRYRSQSISG IPARFSGSGSGTDFT LTISSLEPEDFAVYYCQQSGSWPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASW CLLNNFYPR EAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLS SPVTKSF NRGEC

Dinutuximab HC (knob, crossed) [S228P.T366W] (SEQ ID NO:31) EVQLLQSGPELEKPGASVMISCKASGSSFTGYNMNWVRQNIGKSLEWIGAIDPYYGGTSY NQKFKGRA

TLTVDKSSSTAYMHLKSLTSEDSAVYYCVSGMEYWGQGTSVTVSSASVAAPSVFIFP PSDEQLKSGTAS

WCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH KVYACEVTH

QGLSSPVTKSFNRGECYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCV WDVSQEDPEVQF

NWYVDGVEVHNAKTKPREEQFNSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIE KTISKAKGQ

PREPQVYTLPPSQEEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSRLT

VDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

Dinutuximab LC (crossed) (SEQ ID NO:32)

EIVMTQSPATLSVSPGERATLSCRSSQSLVHRNGNTYLHWYLQKPGQSPKLLIHKVS NRFSGVPDRFSG

SGSGTDFTLKISRVEAEDLGVYFCSQSTHVPPLTFGAGTKLELKSSASTKGPSVFPL APCSRSTSESTAAL

GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTKTYTC NVDHKPSNTKV DKRVESK

CrossMab KIH IgG4 variant 2 with normal dinutuximab Fab and crossed etaracizumab Fab (4 different chains)

Dinutuximab HC (hole) [S228P.T366S.T368A.Y407V] (SEQ ID NO:33)

EVQLLQSGPELEKPGASVMISCKASGSSFTGYNMNWVRQNIGKSLEWIGAIDPYYGG TSYNQKFKGRA

TLTVDKSSSTAYMHLKSLTSEDSAVYYCVSGMEYWGQGTSVTVSSASTKGPSVFPLA PCSRSTSESTAA

LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTKTYT CNVDHKPSNTK

VDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSQEDP EVQFNWYVDGV

EVHNAKTKPREEQFNSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAK GQPREPQVYT

LPPSQEEMTKNQVSLSCLAKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLV SRLTVDKSRWQE GNVFSCSVMHEALHNHYTQKSLSLSLGK

Dinutuximab LC (normal) (SEQ ID NO: 34)

EIVMTQSPATLSVSPGERATLSCRSSQSLVHRNGNTYLHWYLQKPGQSPKLLIHKVS NRFSGVPDRFSG

SGSGTDFTLKISRVEAEDLGVYFCSQSTHVPPLTFGAGTKLELKRTVAAPSVFIFPP SDEQLKSGTASW

CLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHK VYACEVTHQG LSSPVTKSFNRGEC

Etaracizumab HC (knob, crossed) [S228P.T366W] (SEQ ID NO:35)

QVQLVESGGGWQPGRSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVAKVSSGGGST YYLDTVQG

RFTISRDNSKNTLYLQMNSLRAEDTAVYYCARHLHGSFASWGQGTTVTVSSASVAAP SVFIFPPSDEQL

KSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSK ADYEKHKVY

ACEVTHQGLSSPVTKSFNRGECYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRT PEVTCWVDVSQE

DPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRWSVLTVLHQDWLNGKEYKCKVSNKG LPSSIEKTI SKAKGQPREPQVYTLPPSQEEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSF

FLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

Etaracizumab LC (crossed) (SEQ ID NO:36)

EIVLTQSPATLSLSPGERATLSCQASQSISNFLHWYQQRPGQAPRLLIRYRSQSISG IPARFSGSGSGTDFT

LTISSLEPEDFAVYYCQQSGSWPLTFGGGTKVEIKSSASTKGPSVFPLAPCSRSTSE STAALGCLVKDYFP

EPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTKTYTCNVDHKPSNT KVDKRVESK