WO2020018556A1 - Method of treating multiple myeloma - Google Patents

Abstract

The disclosure provides a method of treating multiple myeloma, the method comprising administering to a subject in need thereof a heterodimeric antibody that binds CD3 and CD38.

Description

METHOD OF TREATING MULTIPLE MYELOMA

CROSS REFERENNCE TO RELATED APPLICATION AND INCORPORATION BY

REFERENCE

[0001] This application claims priority to U.S. Provisional Patent Application No.

62/698,675, filed July 16, 2018, the contents of which are hereby incorporated by reference.

[0002] This application incorporates by reference International Patent Publication No. WO 2016/086196, filed on November 25, 2015; U.S. Patent Publication No. 20160215063, filed on November 25, 2015; International Patent Publication No. WO 2017/091656, filed on

November 23, 2016; and U.S. Patent No. U.S. Patent No. 9,822,186, filed on March 30, 2015, which are expressly incorporated herein by reference in their entirety, with particular reference to the figures, legends and claims therein.

[0003] Incorporated by reference in its entirety is a computer-readable nucleotide/amino acid sequence listing submitted concurrently herewith and identified as follows: ASCII (text) file named "52589_Seqlisting.txt", 707,436 bytes created July 15, 2019.

BACKGROUND

[0004] Multiple myeloma is a neoplastic plasma-cell disorder characterized by clonal proliferation of malignant plasma cells in the bone marrow (BM) microenvironment, monoclonal protein in the blood or urine and associated organ dysfunction. Multiple myeloma accounts for 1-2% of all new cancer diagnoses and approximately 20% of all deaths from blood malignancies. The disease is slightly more common in males and African- Americans. Multiple myeloma remains an incurable cancer, although recent improved understanding of pathogenesis of myeloma has led to the development of new treatments and improved survival.

[0005] The diagnosis of multiple myeloma requires the presence of one or more myeloma defining events (MDE) in addition to evidence of either 10% or more clonal plasma cells on BM examination or a biopsy-proven plasmacytoma. MDE include so-called CRAB

(hypercalcemia, renal failure, anemia, or lytic bone lesions) features as well as three specific biomarkers: clonal BM plasma cells > 60%, serum free light chain (sFLC) ratio > 100

(provided involved sFLC level is > 100 mg/L), and more than 1 focal lesion on magnetic resonance imaging (MRI) (Rajkumar et al, 2011). Several genetic abnormalities that occur in tumor plasma cells play major roles in the pathogenesis of myeloma and determine disease prognosis (Palumbo and Anderson, 2011).

[0006] The uncontrolled growth of myeloma cells has many consequences, including skeletal destruction, BM failure, increased plasma volume and viscosity, suppression of normal immunoglobulin production, and renal impairment (Durie, 2011).

[0007] Symptomatic (active) disease should be treated immediately, whereas asymptomatic (smoldering) myeloma requires only clinical observation, since early treatment with conventional chemotherapy has shown no clear benefit yet. Investigational trials are currently evaluating the ability of immunomodulatory drugs to delay the progression from asymptomatic to symptomatic myeloma. For active myeloma, current data support the initiation of induction therapy regimens including thalidomide, lenalidomide, and/or bortezomib followed by autologous hematopoietic stem-cell transplantation (HSCT) after major disease response for patients who can tolerate auto-HSCT conditioning regimens. Considerations of physiologic age, which may differ from chronologic age, and the presence of coexisting conditions drive decisions of treatment choice and drug dose. For example, less intensive approaches are desirable for patients with significant comorbidities, including cardiopulmonary or hepatic impairment, limiting treatment-related mortality and mitigating risk of treatment interruption.

[0008] Treatment of relapsed/refractory multiple myeloma presents a special therapeutic challenge, due to the heterogeneity of disease at relapse and the absence of clear biological based recommendations regarding the choice of salvage therapies at various time points of disease progression. With increasing recognition of the inherent clonal heterogeneity and genomic instability of the plasma cells influencing both inherent and acquired therapeutic resistance, the identification of the optimal choice and sequence of therapies has become critical. New agents have gained approval by United States (US) Food and Drug

Administration (FDA) for relapsed/refractory myeloma in recent years, adding to the complexity of these decisions, including proteasome inhibitors (cafilzomib and ixazomib), the thalidomide derivative pomalidomide, and the histone deacetylase inhibitor

panobinostat. Other molecularly targeted therapies directed at specific cell signaling pathways, as well as survival and proliferation controls (including PI3K/AKT/mTOR inhibitors, Hsp90 inhibitors, cyclin-dependent kinase inhibitors, kinesin spindle protein inhibitors) are currently in development. Despite advances in the management of multiple myeloma, relapse is inevitable in almost all patients. Recurrence of myeloma is typically more aggressive with each relapse, leading to the development of treatment refractory- disease, which is associated with a shorter survival (Dimopoulos et al, 2015). Thus, additional treatment options are needed.

BRIEF SUMMARY

[0009] The disclosure provides a method of treating multiple myeloma in a subject in need thereof, e.g., a subject suffering from relapsed/refractory multiple myeloma. The method comprises administering to the subject a heterodimeric antibody comprising a) a first monomer comprising a first Fc domain and an anti-CD3 scFv comprising (i) a scFv variable light domain comprising vlCDRl as set forth in SEQ ID NO: 15, vlCDR2 as set forth in SEQ ID NO:16, and vlCDR3 as set forth in SEQ ID NO:17, and (ii) a scFv variable heavy domain comprising vhCDRl as set forth in SEQ ID NO:ll, vhCDR2 as set forth in SEQ ID NO:12, and vhCDR3 as set forth in SEQ ID NO: 13, wherein said scFv is covalently attached to the N-terminus of said Fc domain using a domain linker; b) a second monomer comprising i) an anti-CD38 heavy variable domain comprising vhCDRl as set forth in SEQ ID NO:65, vhCDR2 as set forth in SEQ ID NO:66, and vhCDR3 as set forth in SEQ ID NO:67, and ii) a heavy constant domain comprising a second Fc domain and; and c) a light chain comprising a variable constant domain and an anti-CD38 variable light domain comprising vlCDRl as set forth in SEQ ID NO:69, vlCDR2 as set forth in SEQ ID NO: 70, and vlCDR3 as set forth in SEQ ID NO:71, in a dose of about 0.05 mg to about 200 mg. In various aspects, the dose is about 0.05 mg, 0.15 mg, 0.45 mg, 1.35 mg, 4 mg, 12 mg, 36 mg, 100 mg, or 200 mg, or about 36 mg to about 200 mg.

[0010] Optionally, the method comprises administering two doses of heterodimeric antibody per week to the subject in the first and second weeks of treatment, administering one dose of heterodimeric antibody per week to the subject in the third and fourth weeks of treatment, and administering one dose of heterodimeric antibody every two weeks starting in week 5 through the end of treatment. The treatment period (i.e., the time during which multiple doses of heterodimeric antibody is administered to the subject), in various aspects, comprises about six months to about 12 months, e.g., about eight months. In some aspects of the disclosure, the heterodimeric antibody is administered via intravenous infusion over a period of about 30 minutes to about four hours. For example, optionally, the first dose of heterodimeric antibody is administered over a period of about four hours, the second dose of heterodimeric antibody is administered over a period of about two hours, and all subsequent doses are administered over a period of about 30 minutes. In some aspects wherein multiple doses (i.e., two or more) of the heterodimeric antibody are administered to the subject over the course of a treatment period, the dose of heterodimeric antibody is increased at least once during treatment.

[0011] In some aspects, the method further comprises administering dexamethasone to the subject. The dexamethasone is administered, e.g., intravenously or orally. If administered intravenously, the dexamethasone is optionally administered to the subject within one hour prior to administration of the heterodimeric antibody. Suitable doses of dexamethasone include, but are not limited to, about 8 mg or about 4 mg.

[0012] In various aspects, the subject has been previously treated with an anti-CD38 monospecific antibody, such as daratumumab. In this regard, the subject is administered an anti-CD38 monospecific antibody, and an initial dose of the heterodimeric antibody is administered following a wash-out period sufficient to reduce systemic concentration of the anti-CD38 monospecific antibody to 0.2 pg/ml or less, although this is not required in all aspects of the disclosure. The anti-CD38 monospecific antibody is not administered during the wash-out period (i.e., anti-CD38 monospecific antibody treatment ends, a wash-out period of time is allowed to pass, and then the initial dose of heterodimeric antibody is administered). In some instances, the method comprises ceasing treatment with the anti- CD38 monospecific antibody for at least 12 weeks (e.g., about 13-15 weeks) prior to administering an initial dose of the heterodimeric antibody (i.e., the subject is administered an anti-CD38 monospecific antibody, and is then administered an initial dose of the heterodimeric antibody at a timepoint that is at least 12 weeks after administration of the anti-CD38 monospecific antibody has ceased). In various aspects, the subject was previously treated with a proteasome inhibitor and/or an immunomodulatory drug. [0013] In various embodiments, anti-CD3 scFv comprises a variable heavy domain comprising an amino acid sequence at least 90% identical (e.g., 100% identical) to the amino acid sequence set forth in SEQ ID NO: 10. In various aspects, the anti-CD3 scFv comprises a variable heavy domain comprising an amino acid sequence which comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid substitutions compared to SEQ ID NO: 10, optionally in regions outside the CDRs. Also optionally, the anti-CD3 scFv comprises a variable light domain comprising an amino acid sequence at least 90% identical (e.g., 100% identical) to the amino acid sequence set forth in SEQ ID NO: 14. In various aspects, the anti-CD3 scFv comprises a variable light domain comprising an amino acid sequence which comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid substitutions compared to SEQ ID NO: 14, optionally in regions outside the CDRs. For example, in various embodiments, the anti-CD3 scFv comprises an amino acid sequence at least 90% identical (e.g., 100% identical) to the amino acid sequence set forth in SEQ ID NO: 18. In various aspects, the anti-CD3 scFv comprises an amino acid sequence which comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid substitutions compared to SEQ ID NO: 18, optionally in regions outside the CDRs. In some aspects of the disclosure, the scFv domain linker is a charged linker.

Optionally, the anti-CD38 variable light domain comprises an amino acid sequence at least 90% identical (e.g., 100% identical) to the amino acid sequence set forth in SEQ ID NO: 68 and/or the anti-CD38 heavy variable domain comprises an amino acid sequence at least 90% identical (e.g., 100% identical) to the amino acid sequence set forth in SEQ ID NO: 64. For instance, , the anti-CD38 variable light domain comprises an amino acid sequence comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid substitutions compared to SEQ ID NO: 68, and/or the anti-CD38 heavy variable domain comprises an amino acid sequence comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid substitutions compared SEQ ID NO: 64. Optionally, the substitutions are outside the CDRs. For example, in various aspects, the first monomer comprises an amino acid sequence at least 90% identical (e.g., 100% identical) to the amino acid sequence set forth in SEQ ID NO: 335 and/or the second monomer comprises an amino acid sequence at least 90% identical (e.g., 100% identical) to the amino acid sequence set forth in SEQ ID NO: 82 and/or the light chain comprises an amino acid sequence at least 90% identical (e.g., 100% identical) to the amino acid sequence set forth in SEQ ID NO: 84. In some embodiments, the first Fc domain and the second Fc domain of the heterodimeric antibody comprises one or more mutations that reduce homodimerization. In some embodiments, the first Fc domain and said second Fc domain comprise a set of variants selected from the group consisting of S364K/E357Q : L368D/K370S; L368D/K370S : S364K; L368E/K370S : S364K; T411T/E360E/Q362E : D401K; L368D/K370S : S364K/E357L and K370S : S364K/E357Q, with S364K/E357Q : L368D/K370S representing a preferred embodiment. In other aspects, said first and second Fc domains comprise the amino acid substitutions E233P/L234V/L235A/G236del/S267K. Optionally, the heavy chain constant domain comprises the amino acid substitutions

N208D/Q295E/N384D/Q418E/N421D.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figures 1 A and IB depict several formats of heterodimeric antibodies. Two forms of the "bottle opener" format are depicted, one with the anti-CD3 antigen binding domain comprising a scFv and the anti-CD38 antigen binding domain comprising a Fab, and one with these reversed. The mAb-Fv, mAb-scFv, Central-scFv and Central-Fv formats are all shown. In addition, "one-armed" formats, where one monomer just comprises an Fc domain, are shown, both a one arm Central-scFv and a one arm Central-Fv. A dual scFv format is also shown.

[0015] Figure 2 depicts the sequences of the "High-Int #l"Anti-CD3_H1.32_L1.47 construct, including the variable heavy and light domains (CDRs underlined), as well as the individual vl and vhCDRs, as well as an scFv construct with a charged linker (double underlined). As is true of all the sequences depicted in the Figures, this charged linker may be replaced by an uncharged linker or a different charged linker, as needed.

[0016] Figure 3 depicts the sequences of the intermediate CD38: OKT10 H1F1.24 construct, including the variable heavy and light domains (CDRs underlined), as well as the individual vl and vhCDRs, as well as an scFv construct with a charged linker (double underlined).

[0017] Figure 4 depicts the sequences of the Fow CD38: OKT10 H1F1 construct, including the variable heavy and light domains (CDRs underlined), as well as the individual vl and vhCDRs, as well as an scFv construct with a charged linker (double underlined).

[0018] Figure 5 depicts the sequences of XENP18971. [0019] Figure 6 depicts the sequences of XENP18969.

[0020] Figure 7 depicts the sequence of human CD3 e (SEQ ID NO: 130).

[0021] Figure 8 depicts the full length (SEQ ID NO:131) and extracellular domain (ECD;

SEQ ID NO:132) of the human CD38 protein.

[0022] Figures 9A -9E depict useful pairs of heterodimerization variant sets (including skew and pi variants).

[0023] Figure 10 depicts a list of isosteric variant antibody constant regions and their respective substitutions. pl_(-) indicates lower pi variants, while pl_(+) indicates higher pi variants. These can be optionally and independently combined with other

heterodimerization variants of the invention (and other variant types as well, as outlined herein).

[0024] Figure 11 depicts useful ablation variants that ablate FcyR binding (sometimes referred to as "knock outs" or "KO" variants).

[0025] Figure 12 shows two embodiments of the invention.

[0026] Figures 13A and 13B depict a number of charged scFv linkers that find use in increasing or decreasing the pi of heterodimeric antibodies that utilize one or more scFv as a component. A single prior art scFv linker with a single charge is referenced as "Whitlow", from Whitlow et al., Protein Engineering 6(8):989-995 (1993). It should be noted that this linker was used for reducing aggregation and enhancing proteolytic stability in scFvs.

[0027] Figure 14 depicts a list of engineered heterodimer-skewing Fc variants with heterodimer yields (determined by HPFC-CIEX) and thermal stabilities (determined by DSC). Not determined thermal stability is denoted by "n.d.".

[0028] Figures 15A-15B depict stability-optimized, humanized anti-CD3 variant scFvs. Substitutions are given relative to the H1 F1.4 scFv sequence. Amino acid numbering is Kabat numbering.

[0029] Figures 16A-16B. Amino acid sequences of stability-optimized, humanized anti-CD3 variant scFvs. CDRs are underlined. For each heavy chain/light chain combination, four sequences are listed: (i) scFv with C-terminal 6xHis tag, (ii) scFv alone, (iii) VH alone, (iv) VL alone.

[0030] Figure 17 depicts the sequences of XENP18971.

[0031] Figure 18 depicts the sequences of XENP18969.

[0032] Figure 19 shows a matrix of possible combinations of embodiments. An "A" means that the CDRs of the referenced CD3 sequences can be combined with the CDRs of CD38 construct on the left hand side. That is, for example for the top left hand cell, the vhCDRs from the variable heavy chain CD3 HI.30 sequence and the vlCDRs from the variable light chain of CD3 LI.47 sequence can be combined with the vhCDRs from the CD38 OKT10 HI.77 sequence and the vlCDRs from the

OKT10L1.24 sequence. A "B" means that the CDRs from the CD3 constructs can be combined with the variable heavy and light domains from the CD38 construct. That is, for example for the top left hand cell, the vhCDRs from the variable heavy chain CD3 HI.30 sequence and the vlCDRs from the variable light chain of CD3 LI.47 sequence can be combined with the variable heavy domain CD38 OKT10 HI.77 sequence and the OKT10L1.24 sequence. A "C" is reversed, such that the variable heavy domain and variable light domain from the CD3 sequences are used with the CDRs of theCD38 sequences. A "D" is where both the variable heavy and variable light chains from each are combined. An "E" is where the scFv of the CD3 is used with the CDRs of the CD38 antigen binding domain construct, and an "E" is where the scFv of the CD3 is used with the variable heavy and variable light domains of the CD38 antigen binding domain.

[0033] Figure 20 depicts the sequences of Ab-A.

[0034] Figure 21 includes two graphs illustrating the response of pretreatment of dexamethasone then exposure to Ab-A described in the Example. The left graph correlates cytotoxicity (%) (Y-axis) with concentration of Ab-A (ng/ml). Pretreatment with

dexamethasone had minimal effect on EC50 of the heterodimeric antibody. The right graph correlates cytokine production (IFNgamma, pg/ml) (y-axis) with concentration of Ab-A (ng/ml). Pretreatment resulted in an 85% decrease in cytokine release.

[0035] Figure 22 is a table associating various CDR sequences, variable region sequences, heavy and light chain sequences, scFv sequences, backbone sequences, etc. with sequence identifiers set forth in the sequence listing accompanying the instant application. Regarding the several bottle opener format backbones noted (SEQ ID NOs: 347-354), the sequences are provided without the Fv sequences (e.g., the scFv and the vh and vl for the Fab side). As will be appreciated by those in the art and outlined below, these sequences can be used with any vh and vl pairs outlined herein, with one monomer including a scFv (optionally including a charged scFv linker) and the other monomer including the Fab sequences (e.g., a vh attached to the "Fab side heavy chain" and a vl attached to the "constant light chain").

The scFv can be anti-CD3 or anti-CD38, with the Fab being the other. ("Fab" referring to the portion that comprises the VH, CHI, VL, and CL immunoglobulin domains.) That is, any Lv sequences outlined herein for CD3 and CD38 can be incorporated into these backbones in any combination.

DETAILED DESCRIPTION

[0036] The disclosure provides a method of treating multiple myeloma in a subject in need thereof. The method employs a heterodimeric antibody that co-engages CD3 and CD38 in such a manner so as to transiently connect malignant cells with T cells, thereby inducing T cell mediated killing of the bound malignant cell. CD3-targeting approaches have shown considerable promise, yet a common side effect of such therapies is the associated production of cytokines, often leading to toxic cytokine release syndrome. Because the anti- CD3 binding domain of the bispecific antibody engages all T cells, the high cytokine- producing CD4 T cell subset is recruited. Moreover, the CD4 T cell subset includes regulatory T cells, whose recruitment and expansion can potentially lead to immune suppression and have a negative impact on long-term tumor suppression. One such possible way to reduce cytokine production and possibly reduce the activation of CD4 T cells is by reducing the affinity of the anti-CD3 domain for CD3. However, too great of a reduction in affinity may lead to reduced efficacy of a therapeutic comprising the anti-CD3 domain. The affinity for CD38 of a bispecific antibody also has an effect on the efficacy of the antibody in targeting cells expressing CD38. The method described herein utilizes a heterodimeric antibody that binds CD3 and CD38 in such a manner so as to maximize destruction of target cells while reducing unwanted side effects (e.g., uncontrolled cytokine release).

[0037] The heterodimeric antibodies described herein use different monomers which contain amino acid substitutions that "skew" formation of heterodimers over homodimers, as is more fully outlined below, coupled with "pi variants" that allow simple purification of the heterodimers away from the homodimers. The heterodimeric antibody optionally comprises engineered or variant Fc domains that self-assemble in production cells to produce heterodimeric proteins.

[0038] Various aspects of the method are described below. The use of section headings are merely for the convenience of reading, and not intended to be limiting per se. The entire document is intended to be viewed as a unified disclosure, and it should be understood that all combinations of features described herein are contemplated.

Heterodimeric antibody

[0039] The method comprises administering to the subject in need thereof a heterodimeric antibody comprising a) a first monomer comprising a first Fc domain and an anti-CD3 scFv. An scFv comprises a variable heavy chain, an scFv linker, and a variable light domain.

Optionally, the C-terminus of the variable light chain is attached to the N-terminus of the scFv linker, the C-terminus of which is attached to the N-terminus of a variable heavy chain (N-vh-linker-vl-C), although the configuration can be switched (N-vl-linker-vh-C). Thus, specifically included in the depiction and description of scFvs are the scFvs in either orientation. In various aspects, the scFv domain linker is a charged linker. A number of suitable scFv linkers can be used and many are set forth in the Figures. Charged scFv linkers may be employed to facilitate the separation in pi between a first and a second monomer. That is, by incorporating a charged scFv linker, either positive or negative (or both, in the case of scaffolds that use scFvs on different monomers), this allows the monomer comprising the charged linker to alter the pi without making further changes in the Fc domains. [0040] The scFv is covalently attached to the N-terminus of the Fc domain using a domain linker. A "domain linker" links any two domains as outlined herein together. If desired, charged domain linkers can be used. Charged domain linkers can, e.g., increase the pi separation of the monomers of the disclosure as well, and thus those included in the Figures can be used in any embodiment herein where a linker is utilized.

[0041] A linker peptide may predominantly include the following amino acid residues: Gly, Ser, Ala, or Thr. The linker peptide should have a length that is adequate to link two molecules in such a way that they assume the correct conformation relative to one another so that they retain the desired activity. In one embodiment, the linker is from about 1 to 50 amino acids in length, preferably about 1 to 30 amino acids in length. In one embodiment, linkers of 1 to 20 amino acids in length may be used, with from about 5 to about 10 amino acids finding use in some embodiments. Useful linkers include glycine-serine polymers, including for example (GS)n, (GSGGS)n (SEQ ID NO:332), (GGGGS)n (SEQ ID NO:333), and (GGGS)n (SEQ ID NO:334), where n is an integer of at least one (and generally from 3 to 4), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers. Alternatively, a variety of nonproteinaceous polymers, including but not limited to polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol and polypropylene glycol, may find use as linkers, that is may find use as linkers.

[0042] Other linker sequences may include any sequence of any length of CL/CHI domain but not all residues of CL/CHI domain; for example the first 5-12 amino acid residues of the CL/CHI domains. Linkers can be derived from immunoglobulin light chain, for example CK or Cl. Linkers can be derived from immunoglobulin heavy chains of any isotype, including for example Cyl, Cy2, Cy3, Cy4, Cal, Ca2, C5, Ce, and Cp. Linker sequences may also be derived from other proteins such as Ig-like proteins (e.g. TCR, FcR, KIR), hinge region- derived sequences, and other natural sequences from other proteins.

[0043] The anti-CD3 scFv comprises (i) a scFv variable light domain comprising vlCDRl as set forth in SEQ ID NO: 15, vlCDR2 as set forth in SEQ ID NO: 16, and vlCDR3 as set forth in SEQ ID NO: 17, and (ii) a scFv variable heavy domain comprising vhCDRl as set forth in SEQ ID NO: 11, vhCDR2 as set forth in SEQ ID NO:12, and vhCDR3 as set forth in SEQ ID NO: 13. Optionally, the anti-CD3 scFv comprises a variable heavy domain comprising an amino acid sequence at least 90% identical (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO: 10. In various aspects, the anti-CD3 scFv comprises a variable heavy domain comprising an amino acid sequence which comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid

substitutions compared to SEQ ID NO: 10, optionally in regions outside the CDRs. Also optionally, the anti-CD3 scFv comprises a variable light domain comprising an amino acid sequence at least 90% identical (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO: 14. In various aspects, the anti-CD3 scFv comprises a variable light domain comprising an amino acid sequence which comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid substitutions compared to SEQ ID NO: 14, optionally in regions outside the CDRs. In this regard, the anti-CD3 scFv, in various embodiments, comprises a variable heavy domain of SEQ ID NO: 10 and a variable light domain of SEQ ID NO: 14. Optionally, the variable heavy and variable light domains are linked by an scFv domain linker comprising the sequence GKPGSGKPGSGKPGSGKPGS (SEQ ID NO: 158). In this regard, the anti-CD3 scFv comprises, in various embodiments, an amino acid sequence at least 90% identical (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO: 18. In various aspects, the anti-CD3 scFv comprises an amino acid sequence which comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid substitutions compared to SEQ ID NO: 18. In various aspects, the sequence variation giving rise to less than 100% percent identity to a reference sequence represents modifications outside the CDR sequences. In various aspects, the scFv comprises sequences set forth herein as belonging to Anti-CD3_H1.32_L1.47.

[0044] "Fc" or "Fc region" or "Fc domain" refers to the polypeptide comprising the constant region of an antibody excluding the first constant region immunoglobulin domain and in some cases, part of the hinge. Thus Fc domain refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG, the last three constant region

immunoglobulin domains of IgE and IgM, and the flexible hinge N-terminal to these domains. For IgA and IgM, Fc may include the J chain. For IgG, the Fc domain comprises immunoglobulin domains Cy2 and Cy3 (Cy2 and Cy3) and the lower hinge region between Cyl (Cyl) and Cy2 (Cy2). The heterodimeric antibody is preferably an IgG antibody (which includes several subclasses, including, but not limited to IgGl, IgG2, IgG3, and IgG4). Although the boundaries of the Fc region may vary, the human IgG heavy chain Fc region is usually defined to include residues C226 or P230 to its carboxyl-terminus, wherein the numbering is according to the EU index as in Kabat. In some embodiments, amino acid modifications are made to the Fc region, for example, to alter binding to one or more FcyR receptors or to the FcRn receptor.

[0045] In various aspects, the first monomer (i.e., the first Fc domain and the anti-CD3 scFv) comprises an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO:335 (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 335). In various aspects, the sequence variation giving rise to less than 100% percent identity to a reference sequence represents modifications outside the CDR sequences.

[0046] The heterodimeric antibody of the method further comprises b) a second monomer comprising i) an anti-CD38 heavy variable domain and ii) a heavy constant domain comprising a second Fc domain. The anti-CD38 heavy variable domain comprises the following CDR sequences: variable heavy (vh) CDR1 as set forth in SEQ ID NO:65, vhCDR2 as set forth in SEQ ID NO:66, and vhCDR3 as set forth in SEQ ID NO:67. Optionally, the anti-CD38 heavy variable domain comprises an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO:64 (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 64). In various aspects, the anti-CD38 variable heavy domain comprises an amino acid sequence which comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid substitutions compared to SEQ ID NO: 64. In various aspects, the second monomer (i.e., the anti-CD38 variable heavy domain and heavy constant domain comprising a second Fc domain) comprises an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO:82 (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 82). In various aspects, the sequence variation giving rise to less than 100% percent identity to a reference sequence represents modifications outside the CDR sequences.

[0047] The heterodimeric antibody further comprises c) a light chain comprising a variable constant domain and an anti-CD38 variable light (vl) domain. The anti-CD38 variable light domain comprises the following CDRs: vlCDRl as set forth in SEQ ID NO:69, vlCDR2 as set forth in SEQ ID NO: 70, and vlCDR3 as set forth in SEQ ID NO: 71. Optionally, the anti- CD38 variable light domain comprises an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO:68 (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 68). In various aspects, the anti-CD38 variable light domain comprises an amino acid sequence which comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid substitutions compared to SEQ ID NO: 68. In some embodiments, the light chain (comprising the variable constant domain and the anti-CD38 variable light domain) comprises an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO:84 (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 84). In various aspects, the sequence variation giving rise to less than 100% percent identity to a reference sequence represents modifications outside the CDR sequences.

[0048] In a preferred embodiment, the heterodimeric antibody comprises a first monomer comprising an anti-CD3 scFv comprising an anti-CD3 variable light domain comprising the amino acid sequence of SEQ ID NO: 14 and an anti-CD3 variable heavy domain comprising the amino acid sequence of SEQ ID NO: 10, a second monomer comprising an anti-CD38 variable heavy domain comprising the amino acid sequence of SEQ ID NO: 64, and a light chain comprising a variable light domain comprising the amino acid sequence of SEQ ID NO: 68. For example, in one embodiment, the heterodimeric antibody comprises a first monomer comprising the amino acid sequence of SEQ ID NO: 335, a second monomer comprising the amino acid sequence of SEQ ID NO: 82, and a light chain comprising the amino acid sequence of SEQ ID NO: 84.

[0049] The heterodimeric antibody adopts the structure termed "bottle opener" in Figure 1A. One heavy chain of the "bottle opener" format contains the scFv and the other heavy chain is a "regular" Fab format, comprising a variable heavy chain and a light chain. The two chains are brought together by the use of amino acid variants in the constant regions (e.g., the Fc domain, the CHI domain and/or the hinge region) that promote the formation of heterodimeric antibodies. There are several distinct advantages to the "bottle opener" format. Antibody analogs relying on two scFv constructs often have stability and aggregation problems, which is alleviated in the present disclosure by the addition of a "regular" heavy and light chain pairing. In addition, as opposed to formats that rely on two heavy chains and two light chains, there is no issue with the incorrect pairing of heavy and light chains (e.g., heavy 1 pairing with light 2, etc.).

[0050] The heterodimeric antibody includes, in various aspects, modifications as compared wild-type antibody domain sequences to promote heterodimeric antibody formation (i.e., reduce homodimerization), adjust antibody functionality, etc. Modifications generally are focused in the Fc domain (although this is not required). Modifications are referenced by the amino acid position of the substitution, deletion, or insertion with respect to the native sequence. For example, N434S or 434S is an Fc domain substitution of serine at position 434 relative to the parent Fc polypeptide, wherein the numbering is according to the EU index. Likewise, M428L/N434S defines an Fc modification having substitutions M428L and N434S relative to the parent Fc polypeptide. The identity of the wild-type amino acid may be unspecified, in which case the aforementioned variant is referred to as 428L/434S. The order in which substitutions are provided is arbitrary, that is to say that, for example, 428L/434S is the same as M428L/N434S, and so on. For all positions discussed that relate to antibodies, unless otherwise noted, amino acid position numbering is according to the EU index. The EU index or EU index as in Kabat or EU numbering scheme refers to the numbering of the EU antibody (Edelman et al., 1969, Proc Natl Acad Sci USA 63:78-85, hereby entirely incorporated by reference). The modification can be an addition, deletion, or substitution. Substitutions can include naturally occurring amino acids and, in some cases, synthetic amino acids. Examples include U.S. Patent No. 6,586,207; International Patent Publication Nos. WO 98/48032; WO 03/073238; US2004-0214988A1; WO 05/35727A2; WO 05/74524A2; J. W. Chin et al., (2002), Journal of the American Chemical Society 124:9026-9027; J. W. Chin, & P. G. Schultz, (2002), ChemBioChem 11:1135-1137; J. W. Chin, et al., (2002), PICAS United States of America 99:11020-11024; and, L. Wang, & P. G. Schultz, (2002), Chem. 1-10, all entirely incorporated by reference.

[0051] There are a number of mechanisms that can be used to generate the heterodimeric protein. Amino acid variants that lead to the production of heterodimers are referred to as "heterodimerization variants." Heterodimerization variants can include steric variants (e.g., the "knobs and holes" or "skew" variants described below and the "charge pairs" variants described below) as well as "pi variants," which allow purification of homodimers away from heterodimers. As is generally described in International Patent Publication No.

WO2014/145806, hereby incorporated by reference in its entirety and specifically for the discussion of "heterodimerization variants," useful mechanisms for heterodimerization include "knobs and holes" ("KIH"; sometimes herein as "skew" variants), "electrostatic steering" or "charge pairs" as described in WO2014/145806, pi variants as described in WO2014/145806, and general additional Fc variants as outlined in WO2014/145806 and herein.

[0052] There are several basic mechanisms that can lead to ease of purifying heterodimeric antibodies; one relies on the use of pi variants, such that each monomer has a different pi, thus allowing the isoelectric purification of A- A, A-B and B-B dimeric proteins.

Alternatively, some scaffold formats, such as the "bottle opener" format, also allows separation on the basis of size. It is also possible to "skew" the formation of heterodimers over homodimers. Thus, a combination of steric heterodimerization variants and pi or charge pair variants find particular use in the invention. pi (Isoelectric point) Variants

[0053] For pi variants, amino acid modifications can be introduced into one or both of the monomer polypeptides; that is, the pi of one of the monomers (referred to herein for simplicity as "monomer A") can be engineered away from monomer B, or both monomer A and B change be changed, with the pi of monomer A increasing and the pi of monomer B decreasing. The pi changes of either or both monomers can be done by removing or adding a charged residue (e.g., a neutral amino acid is replaced by a positively or negatively charged amino acid residue, e.g., glycine to glutamic acid), changing a charged residue from positive or negative to the opposite charge (aspartic acid to lysine) or changing a charged residue to a neutral residue (e.g., loss of a charge; lysine to serine). A number of these variants are shown in the Figures. These of modifications create a sufficient change in pi in at least one of the monomers such that heterodimers can be separated from homodimers. As will be appreciated by those in the art, this can be achieved by using a "wild type" heavy chain constant region and a variant region that has been engineered to either increase or decrease it's pi (wt A-+B or wt A - -B), or by increasing one region and decreasing the other region (A+ -B- or A- B+).

[0054] Thus, in various aspects, the heterodimeric antibody comprises one or more modifications in the constant region(s) to alter the isoelectric point (pi) of at least one, if not both, of the monomers of a heterodimeric protein to form "pi antibodies" by incorporating amino acid substitutions ("pi variants" or "pi substitutions") into one or both of the monomers. The separation of the heterodimers from the two homodimers can be accomplished if the pis of the two monomers differ by as little as 0.1 pH unit, with 0.2, 0.3, 0.4 and 0.5 or greater all being suitable.

[0055] The number of pi variants to be included on each or both monomer(s) to achieve good separation will depend in part on the starting pi of the components, for example, the starting pi of the anti-CD3 scFv and anti-CD38 Fab. That is, to determine which monomer to engineer or in which "direction" (e.g. more positive or more negative), the Fv sequences of the two domains are calculated and a decision is made from there. Different Fvs will have different starting pis which can be exploited. In some embodiments, the change in pi is calculated on the basis of the variant heavy chain constant domain, using the chart in the Figure 19 of U.S. Patent Publication No. 2014/0370013. Alternatively, the pi of each monomer can be compared. In general, the pis are engineered to result in a total pi difference of each monomer of at least about 0.1 logs, with 0.2 to 0.5 being preferred.

[0056] Preferred combinations of pi variants are shown in Figure 10. These changes are shown relative to IgGl, but all isotypes can be altered this way, as well as isotype hybrids.

In the case where the heavy chain constant domain is from IgG2-4, R133E and R133Q can also be used.

[0057] In one embodiment, the Fab monomer (the negative side) comprises the substitutions 208D/295E/384D/418E/421D (N208D/Q295E/N384D/Q418E/N421D when relative to human IgGl) and the scFv monomer (the positive side) comprises a positively charged scFv linker, including (GKPGS)4.

[0058] Modifications to adjust pi also can be made in the light chain. Amino acid substitutions for lowering the pi of the light chain include, but are not limited to, K126E, K126Q, K145E, K145Q, N152D, S156E, K169E, S202E, K207E and adding peptide DEDE at the C-terminus of the light chain. Changes in this category based on the constant lambda light chain include one or more substitutions at R108Q, Q124E, K126Q, N138D, K145T and Q199E. In addition, increasing the pi of the light chains can also be done.

Skew/Steric Variants

[0059] There are a number of suitable pairs of sets of heterodimerization skew variants. These variants come in "pairs" of "sets." That is, one set of the pair is incorporated into the first monomer and the other set of the pair is incorporated into the second monomer. It should be noted that these sets do not necessarily behave as "knobs in holes" variants, with a one-to-one correspondence between a residue on one monomer and a residue on the other; that is, these pairs of sets form an interface between the two monomers that encourages heterodimer formation and discourages homodimer formation, allowing the percentage of heterodimers that spontaneously form under biological conditions to be over 90%, rather than the expected 50% (25 % homodimer A/A:50% heterodimer A/B:25% homodimer B/B).

[0060] In some embodiments, the formation of heterodimers is facilitated by the addition of steric variants. That is, by changing amino acids in each heavy chain, different heavy chains are more likely to associate to form the heterodimeric structure than to form homodimers with the same Fc amino acid sequences. Suitable examples of steric variants are included in Figure 9.

[0061] One mechanism is generally referred to in the art as "knobs and holes," referring to amino acid engineering that creates steric influences to favor heterodimeric formation and disfavor homodimeric formation, can also optionally be used. This is further described in U.S. Patent Publication No. 20130205756, Ridgway et al., Protein Engineering 9(7):617 (1996); Atwell et al., J. Mol. Biol. 1997 270:26; U.S. Patent No. 8,216,805, all of which are hereby incorporated by reference in their entirety. The Figures identify a number of "monomer A - monomer B" pairs that rely on "knobs and holes." In addition, as described in Merchant et al., Nature Biotech. 16:677 (1998), these "knobs and hole" mutations can be combined with disulfide bonds to skew formation to heterodimerization.

[0062] An additional mechanism that finds use in the generation of heterodimers is sometimes referred to as "electrostatic steering" as described in Gunasekaran et al., J. Biol. Chem. 285(25):19637 (2010), hereby incorporated by reference in its entirety. This is sometimes referred to herein as "charge pairs." In this embodiment, electrostatics are used to skew the formation towards heterodimerization. As those in the art will appreciate, these may also have an effect on pi, and thus on purification, and thus could in some cases also be considered pi variants. However, as these were generated to force heterodimerization and were not used as purification tools, they are classified as "steric variants." These include, but are not limited to, D221E/P228E/L368E paired with D221R/P228R/K409R (i.e., these are monomer corresponding sets) and C220E/P228E/368E paired with

C220R/E224R/P228R/K409R.

[0063] Additional monomer A and monomer B variants that can be combined with other variants, optionally and independently in any amount, such as pi variants outlined herein or other steric variants that are shown in Figure 37 of U.S. Patent Publication No. 2012/0149876, the figure and legend and SEQ ID NOs of which are incorporated expressly by reference herein.

[0064] In some embodiments, the steric variants outlined herein can be optionally and independently incorporated with any pi variant (or other variants such as Fc variants, FcRn variants, etc.) into one or both monomers, and can be independently and optionally included or excluded from the proteins of the invention.

[0065] A list of suitable skew variants is found in Figure 9 and Figure 12. Of particular use in many embodiments are the pairs of sets including, but not limited to, S364K/E357Q : L368D/K370S; L368D/K370S : S364K; L368E/K370S : S364K; T411T/E360E/Q362E : D401K; L368D/K370S : S364K/E357L and K370S : S364K/E357Q. In terms of nomenclature, the pair "S364K/E357Q : L368D/K370S" means that one of the monomers has the double variant set S364K/E357Q and the other has the double variant set L368D/K370S.

Additional Fc Variants for Adjusting Functionality

[0066] There are a number of useful Fc amino acid modification that can be made for a variety of reasons, including, but not limited to, altering binding to one or more FcyR receptors, altered binding to FcRn receptors, etc.

[0067] There are a number of useful Fc substitutions that can be made to alter binding to one or more of the FcyR receptors. Substitutions that result in increased binding as well as decreased binding can be useful. For example, it is known that increased binding to FcyRIIIa generally results in increased ADCC (antibody dependent cell-mediated cytotoxicity; the cell-mediated reaction wherein nonspecific cytotoxic cells that express FcyRs recognize bound antibody on a target cell and subsequently cause lysis of the target cell). Similarly, decreased binding to FcyRIIb (an inhibitory receptor) can be beneficial as well in some circumstances. Amino acid substitutions that find use in the present invention include those listed in U.S. Patent Publication Nos. 2006/0024298 (particularly Figure 41), 2006/0121032, 2006/0235208, 2007/0148170, all of which are expressly incorporated herein by reference in their entirety and specifically for the variants disclosed therein. Particular variants that find use include, but are not limited to, 236 A, 239D, 239E, 332E, 332D,

239D/332E, 267D, 267E, 328F, 267E/328F, 236A/332E, 239D/332E/330Y, 239D, 332E/330L,

243 A, 243L, 264A, 264V and 299T.

[0068] In addition, there are additional Fc substitutions that find use in increased binding to the FcRn receptor and increased serum half life, as specifically disclosed in U.S. Patent Publication No. 2009/0163699, hereby incorporated by reference in its entirety, including, but not limited to, 434S, 434A, 428F, 308F, 2591, 428F/434S, 259I/308F, 436I/428F, 4361 or V/434S, 436V/428F and 259I/308F/428F.

[0069] Another category of functional variants are "FcyR ablation variants" or "Fc knock out (FcKO or KO)" variants. For some therapeutic applications, it is desirable to reduce or remove the normal binding of the Fc domain to one or more or all of the Fey receptors (e.g. FcyRl, FcyRIIa, FcyRIIb, FcyRIIIa, etc.) to avoid additional mechanisms of action. That is, for example, particularly in the use of bispecific antibodies that bind CD3 monovalently, it may be desirable to ablate FcyRIIIa binding to eliminate or significantly reduce ADCC activity. Any level of reduction is contemplated (e.g., 50%, 60%, 70%, 80%, 90%, or 100% reduction in binding or activity). Examples of ablation variant modifications are depicted in Figure 11 Figure , and each can be independently and optionally included or excluded, with preferred aspects utilizing ablation variants selected from the group consisting of

G236R/F328R, E233P/F234V/F235A/G236del/S239K, E233P/F234V/F235A/G236del/S267K, E233P/F234V/F235A/G236del/S239K/A327G, E233P/F234V/F235A/G236del/S267K/A327G and E233P/F234V/F235A/G236del. It should be noted that the ablation variants referenced herein ablate FcyR binding but generally not FcRn binding. Additional antibody considerations

[0070] The disclosure contemplates the use of other heterodimeric antibodies in the method. For example, the variable heavy and light sequences, as well as the scFv sequences (and Fab sequences comprising these variable heavy and light sequences) described above can be used in other formats, such as those depicted in Figure 2 of International Patent Publication No. 2014/145806, the Figures, formats and legend of which is expressly incorporated herein by reference, as well as Figures 1A and IB. Further, the amino acid sequences (e.g., CDR sequences, variable light and variable heavy chain sequences, and/or full length heavy and light chain sequences) of CD3-binding regions and CD38-binding regions are provided in the sequence listing provided herewith and summarized in Figure 22. Any combination of the sequences referenced in Figure 22 are contemplated herein so long as the resulting heterodimeric antibody engages both CD3 and CD38. Anti-CD3/anti-CD38 antibodies are further described in reference International Patent Publication No. WO 2016/086196; U.S. Patent Publication No. 20160215063; International Patent Publication No. WO 2017/091656; and U.S. Patent No. U.S. Patent No. 9,822,186, which are incorporated by reference herein in their entirety and particularly with respect to the description of anti-CD3/anti-CD38 antibodies and their amino acid and nucleic acid sequences, sequence listing, and Figures.

[0071] With respect to CD3 binding, the heterodimeric antibody may comprise an anti-CD3 antigen binding domain that has an intermediate or "medium" affinity to CD3 that also bind to CD38. In this regard, the heterodimeric antibody binds to CD3 with an affinity (KD) of about 15-50 nM (e.g., about 16-50 nM, 15-45 nM, about 20-40 nM, about 25-40 nM, or about 30-40 nM), optionally measured using the assays described in U.S. Patent Publication No. 20160215063 and International Patent Publication No. WO 2017/091656, incorporated by reference herein.

[0072] In another aspect, the heterodimeric antibody of the method comprises an anti-CD3 antigen binding domain that is a "strong" or "high affinity" binder to CD3 (e.g., one example are heavy and light variable domains depicted as H1.30_L1.47 (optionally including a charged linker as appropriate)) and also bind to CD38. In various embodiments, the antibody construct binds to CD3 with an affinity (KD) of about 3-15 nM (e.g., 3-10 nM or 4-7 nM), optionally measured using the assays described in U.S. Patent Publication No. 20160215063 and International Patent Publication No. WO 2017/091656, incorporated by reference herein. In other embodiments, the method employs a heterodimeric antibody comprising an anti-CD3 antigen binding domain that is a "lite" or "lower affinity" binder to CD3. In this regard, the heterodimeric antibody optionally binds to CD3 with an affinity (KD) of about 51 nM or more (e.g., 51-100 nM), optionally measured using the assays described in in U.S. Patent Publication No. 20160215063 and International Patent Publication No. WO 2017/091656, incorporated by reference herein.

[0073] The affinity for CD38 of a bispecific antibody also has an effect on the efficacy of the antibody in targeting cells expressing CD38. Bispecific antibodies having "medium" or "low" affinity for CD38 are able to efficiently kill target cells in vitro and in vivo with reduced toxicity profiles. In various embodiments, bispecific antibodies demonstrating "high" affinity for CD38 bind to CD38 with an affinity (KD), e.g., below 1 nM; bispecific antibodies demonstrating "medium" or "intermediate" affinity for CD38 bind CD38 with an affinity (KD) of about, e.g., 1-10 nM (e.g., 2-8 nM or 3-7 nM); bispecific antibodies demonstrating "low" or "lite" affinity for CD38 bind CD38 with an affinity (KD) of about, e.g., 11 nM or more (such as 11-100 nM), all optionally measured using the methods set forth in U.S. Patent Publication No. 20160215063 and International Patent Publication No. WO 2017/091656, incorporated by reference herein.

[0074] Generally, specific binding can be exhibited, for example, by an antibody having a KD for an antigen of at least about 10^-4 M, at least about 10⁵ M, at least about 10⁶ M, at least about 10⁷ M, at least about 10⁸ M, at least about 10⁹ M, alternatively at least about 10¹⁰ M, at least about 10¹¹ M, at least about 10¹² M, or greater, where KD refers to a dissociation rate of a particular antibody-antigen interaction. Typically, an antibody that specifically binds an antigen will have a KD that is 20-, 50-, 100-, 500-, 1000-, 5,000-, 10,000- or more times greater for a control molecule relative to the antigen. Also, specific binding for a particular antigen can be exhibited, for example, by an antibody having a KA or Ka for an antigen or epitope of at least 20-, 50-, 100-, 500-, 1000-, 5,000-, 10,000- or more times greater for the antigen relative to a control, where KA or Ka refers to an association rate of a particular antibody-antigen interaction. [0075] Optionally, the heterodimeric antibody comprises a substitution of the cysteine at position 220 for serine; generally this is on the "scFv monomer" side of the heterodimeric antibody, although it can also be on the "Fab monomer" side, or both, to reduce disulfide formation. Specifically included within the sequences herein are one or both of these cysteines replaced (C220S).

Fragments

[0076] The disclosure also contemplates the use of antibody fragments (distinguished from a full length antibody which constitutes the natural biological form of an antibody, including variable and constant regions, which generally include Fab and Fc domains alongside optional extra antigen binding domains such as scFvs). The antibody fragment contains at least one constant domain which can be engineered to produce heterodimers, such as pi engineering. Other antibody fragments that can be used include fragments that contain one or more of the CHI, CH2, CH3, hinge and CL domains of the invention that have been pi engineered.

Chimeric/Humanized

[0077] The heterodimeric antibody can be a mixture from different species, e.g., a chimeric antibody and/or a humanized antibody. In general, both "chimeric antibodies" and

"humanized antibodies" refer to antibodies that combine regions from more than one species. For example, "chimeric antibodies" traditionally comprise variable region(s) from a mouse (or rat, in some cases) and the constant region(s) from a human. "Humanized antibodies" generally refer to non-human antibodies that have had the variable-domain framework regions swapped for sequences found in human antibodies. Generally, in a humanized antibody, the entire antibody, except the CDRs, is encoded by a polynucleotide of human origin or is identical to such an antibody except within its CDRs. The CDRs, some or all of which are encoded by nucleic acids originating in a non-human organism, are grafted into the beta-sheet framework of a human antibody variable region to create an antibody, the specificity of which is determined by the engrafted CDRs. The creation of such antibodies is described in, e.g., International Patent Publication No. WO 92/11018, Jones,

1986, Nature 321:522-525, and Verhoeyen et ak, 1988, Science 239:1534-1536, all entirely incorporated by reference. "Backmutation" of selected acceptor framework residues to the corresponding donor residues is often required to regain affinity that is lost in the initial grafted construct (U.S. Patent Nos. 5530101; 5585089; 5693761; 5693762; 6180370; 5859205; 5821337; 6054297; and 6407213, all entirely incorporated by reference). The humanized antibody also may comprise at least a portion of an immunoglobulin constant region, typically that of a human immunoglobulin, and thus will typically comprise a human Fc region. Humanized antibodies can also be generated using mice with a genetically engineered immune system. Roque et al., 2004, Biotechnol. Prog. 20:639-654, entirely incorporated by reference. A variety of techniques and methods for humanizing and reshaping non-human antibodies are well known in the art (See Tsurushita & Vasquez, 2004, Humanization of Monoclonal Antibodies, Molecular Biology of B Cells, 533-545, Elsevier Science (USA), and references cited therein, all entirely incorporated by reference).

Humanization methods include but are not limited to methods described in Jones et al.,

1986, Nature 321:522-525; Riechmann et al.,1988; Nature 332:323-329; Verhoeyen et al., 1988, Science, 239:1534-1536; Queen et al., 1989, Proc Natl Acad Sci, USA 86:10029-33; He et al., 1998, J. Immunol. 160: 1029-1035; Carter et al., 1992, Proc Natl Acad Sci USA 89:4285-9,

Presta et al., 1997, Cancer Res. 57(20):4593-9; Gorman et al., 1991, Proc. Natl. Acad. Sci. USA 88:4181-4185; O'Connor et al., 1998, Protein Eng 11:321-8, all entirely incorporated by reference. Humanization or other methods of reducing the immunogenicity of nonhuman antibody variable regions may include resurfacing methods, as described for example in Roguska et al., 1994, Proc. Natl. Acad. Sci. USA 91:969-973, entirely incorporated by reference.

Therapeutic regimen

[0078] The heterodimeric antibody is administered to a subject in need thereof, e.g., a human subject suffering from multiple myeloma, such as relapsed/refractory multiple myeloma. Relapsed myeloma is characterized as a recurrence of disease after prior response. Examples of laboratory and radiological criteria signaling the disease include, but are not limited to, > 25% increase of the serum or urine monoclonal protein (M-protein) or > 25% difference between involved and uninvolved serum free light chains from nadir, respectively, or the development of new plasmacytomas or hypercalciemia. Sonneveld et al., Haematologica. 2016 Apr; 101(4): 396-406. In non-secretory disease patients, relapse is characterized by an increase of the bone marrow plasma cells. A signal for relapsed disease also is characterized by the appearance or reappearance of one or more CRAB criteria or a rapid and consistent biochemical relapse. Refractory myeloma is myeloma that is not responsive to treatment. Relapsed/refractory multiple myeloma refers to the disease which becomes non-responsive or progressive on therapy or within 60 days of the last treatment in patients who previously achieved at least a minimal response on previous therapy.

Sonneveld, supra ; Anderson et al., Leukemia. 2008;22(2):231-239.

[0079] The method of the disclosure comprises administering to the subject a dose of about 0.05 mg to about 200 mg of the heterodimeric antibody. The dose is, in various

embodiments, about 0.5 mg to about 200 mg, about 0.5 to about 150 mg, about 1 mg to about 150 mg, about 10 mg to about 100 mg, about 10 mg to about 200 mg, about 4 mg to about 200 mg, about 12 mg to about 200 mg, about 12 mg to about 100 mg, about 36 mg to about 200 mg, about 36 mg to about 100 mg, or about 100 mg to about 200 mg. In various aspects of the method, the dose administered to the subject is about 0.05 mg, about 0.15 mg, about 0.45 mg, about 1.35 mg, about 4 mg, about 12 mg, about 36 mg, about 100 mg, or about 200 mg.

[0080] In alternative aspects, a single dose of heterodimeric antibody is at least about 0.05 mg, at least about 0.15 mg, at least about 0.45 mg, at least about 1.35 mg, at least about 4 mg, at least about 12 mg, at least about 36 mg, or at least about 100 mg. In various aspects, a single dose of heterodimeric antibody is no more than about 200 mg (e.g., no more than about 100 mg or no more than about 36 mg). It will be appreciated that a single dose may be administered via multiple administrations (i.e., a divided dose), such that the multiple administrations combine to the dose recited herein. For example, multiple administrations (e.g., two or more injections) combine to be at least about 0.05 mg, at least about 0.15 mg, at least about 0.45 mg, at least about 1.35 mg, at least about 4 mg, at least about 12 mg, at least about 36 mg, or at least about 100 mg. In various aspects, the multiple administrations of a single dose of heterodimeric antibody combine to be no more than about 200 mg (e.g., no more than about 100 mg or no more than about 36 mg).

[0081] In various aspects of the method, the dose is adjusted over the course of treatment. For example, the subject is administered an initial dose at one or more administrations, and a higher dose is used in one or more subsequent administrations. Put another way, the disclosure contemplates increasing the dose of heterodimeric antibody at least once over the course of treatment. Alternatively, the dose may be decreased over the course of treatment, such that amount of heterodimeric antibody is reduced as treatment progresses.

[0082] The disclosure contemplates a method wherein multiple (i.e., two or more) doses of the heterodimeric antibody are administered over the course of a treatment period. The individual doses may be administered at any interval, such as once a week, twice a week, three times a week, four times a week, or five times a week. Individual doses may be administered every two weeks, every three weeks, or every four weeks. In other words, in some aspects, a waiting period of at two weeks passes between heterodimeric antibody administrations to the subject. The waiting period between administrations of the doses need not be consistent over the course of the treatment period. In other words, the interval between doses can be adjusted over the course of treatment. In some aspects, the method comprises administering two doses of heterodimeric antibody per week to the subject in the first and second weeks of treatment (i.e., twice a week for weeks 1 and 2), administering one dose of heterodimeric antibody per week to the subject in the third and fourth weeks of treatment (i.e., once a week for weeks 3 and 4), and administering one dose of heterodimeric antibody every two weeks starting in week 5 through the end of treatment (i.e., there is a waiting period of two weeks between doses starting in week 5 through the end of treatment). While not wishing to be bound to any particular theory, the shorter interval between doses for the first administrations (e.g., two doses per week) promotes rapid target cell clearance. Increasing the interval between doses as set forth herein maintains cell clearance while minimizing unwanted side effects associated with immunotherapy.

Alternatively, in various aspects, the method comprises administering one dose of heterodimeric antibody per week for weeks 1-4 of treatment, and optionally administering one dose of the heterodimeric antibody every two weeks starting in week 5 through the end of treatment.

[0083] The multiple doses of heterodimeric antibody are administered over treatment period of, e.g., three months to about 18 months, or about three months to about 12 months, or about three months to about nine months, or about three months to about six months, or about three months to about eight months, or about six months to about 18 months, or about six months to about 12 months, or about eight months to about 12 months, or about six months to about eight months, or about eight months to about 12 months (e.g., about eight months). Optionally, the multiple (i.e., two or more) doses of the heterodimeric antibody are administered over a treatment period of about 12 weeks to about 52 weeks, or about 12 weeks to about 36 weeks, or about 24 weeks to about 32 weeks, with doses administered twice a week, once a week, once every two weeks, or once every four weeks.

[0084] By "treating" multiple myeloma is meant achievement of any positive therapeutic response with respect to the disease. For example, a positive therapeutic response includes one or more of the following improvements in the disease: (1) a reduction in the number of neoplastic cells; (2) an increase in neoplastic cell death; (3) inhibition of neoplastic cell survival; (4) reduction in paraprotein production by tumor cells; (5) inhibition (i.e., slowing to some extent, preferably halting) of tumor growth; (6) an increased patient survival rate; and (7) some relief from one or more symptoms associated with the disease or condition. Tumor response can be assessed for changes in tumor morphology (i.e., overall tumor burden, tumor size, and the like) using screening techniques such as magnetic resonance imaging (MRI) scan, x-radiographic imaging, computed tomographic (CT) scan, bone scan imaging, endoscopy, and tumor biopsy sampling including bone marrow aspiration (BMA) and counting of tumor cells in the circulation. A complete therapeutic response (i.e., absence of clinically detectable disease with normalization of any previously abnormal radiographic studies, bone marrow, and cerebrospinal fluid (CSF) or abnormal monoclonal protein) is not required; any degree of improvement is contemplated. Various additional parameters associated with disease treatment and improvement are set forth in the Examples.

[0085] The heterodimeric antibody may be administered via any suitable means to the subject, e.g., via intravenous, intraarterial, intralymphatic, intrathecal, intracerebral, intraperitoneal, intracerobrospinal, intradermal, subcutaneous, intraarticular, intrasynovial, oral, topical, or inhalation routes. For example, the heterodimeric antibody may be administered via intravenous administration as a bolus or by continuous infusion over a period of time. In various aspects, the method comprises administering the heterodimeric antibody via intravenous infusion over a period of about 30 minutes to about four hours. Optionally, the time for infusion is decreased in subsequent administrations. For example, in one embodiment, the first dose of heterodimeric antibody is administered over a period of about four hours, and subsequent doses are administered over a period of two hours or less. In this regard, the first dose of heterodimeric antibody is optionally administered over a period of about four hours, the second dose of heterodimeric antibody is optionally administered over a period of about two hours, and subsequent doses are optionally administered over a period of about 30 minutes.

[0086] In some instances, the subject has previously been treated for multiple myeloma. For example, the subject may have previously been administered an immunomodulatory drug (thalidomide, lenalidomide, pomalidomide), a proteasome inhibitor (such as pomalidomide, bortezomib, or carfilzomib), dexamethasone, doxorubicin, or combinations thereof.

Optionally, the subject was previously treated with an anti-CD38 monospecific antibody, such as daratumumab (DARZALEX®). In various embodiments, the subject is relapsed or refractory with prior anti-CD38 monospecific antibody treatment. When the patient has been treated with anti-CD38 monospecific antibody, the initial dose of the heterodimeric antibody is preferably administered following a wash-out period sufficient to reduce systemic concentration of the anti-CD38 monospecific antibody to 0.2 pg/ml or less. Put another way, the method comprises a waiting period between the previous administration of anti-CD38 monospecific antibody and administration of the heterodimeric antibody. In various embodiments, the method comprises ceasing treatment with the anti-CD38 monospecific antibody for at least 12 weeks (e.g., about 13 to about 15 weeks) prior to administering an initial dose of the heterodimeric antibody.

Compositions

[0087] Formulations of the heterodimeric antibodies are prepared for storage by mixing an antibody having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. [1980]), in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids;

antioxidants including ascorbic acid and methionine; preservatives (such as

octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine;

monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

[0088] The formulation may also contain more than one active agent, preferably one or more active agents that do not adversely affect each other. For example, it may be desirable to provide antibodies with other specificities. Alternatively, or in addition, the composition may comprise a cytotoxic agent, cytokine, growth inhibitory agent and/or small molecule antagonist. Such molecules are suitably present in combination in amounts that are effective for the purpose intended.

Co-therapy

[0089] Optionally, the heterodimeric antibody is part of a therapeutic regimen that comprises administration of one or more other therapeutic agents, radiation therapy, stem cell transplantation, and the like.

[0090] The method of the disclosure optionally further comprises administering

dexamethasone to the subject. The dexamethasone may be administered by any route, such as the routes described here. Preferably, the dexamethasone is administered intravenously or orally. When the dexamethasone is administered intravenously, it is optionally administered to the subject within one hour prior to administration of the heterodimeric antibody. The dexamethasone is optionally administered in an amount of about 8 mg or about 4 mg.

[0091] In various embodiments, the method of the disclosure further comprises

administering a chemotherapeutic agent. Non-limiting examples of DNA damaging chemotherapeutic agents include topoisomerase I inhibitors (e.g., irinotecan, topotecan, camptothecin and analogs or metabolites thereof, and doxorubicin); topoisomerase II inhibitors (e.g., etoposide, teniposide, and daunorubicin); alkylating agents (e.g., melphalan, chlorambucil, busulfan, thiotepa, ifosfamide, carmustine, lomustine, semustine,

streptozocin, decarbazine, methotrexate, mitomycin C, and cyclophosphamide); DNA intercalators (e.g., cisplatin, oxaliplatin, and carboplatin); DNA intercalators and free radical generators such as bleomycin; and nucleoside mimetics (e.g., 5-fluorouracil, capecitibine, gemcitabine, fludarabine, cytarabine, mercaptopurine, thioguanine, pentostatin, and hydroxyurea).

[0092] Chemotherapeutic agents that disrupt cell replication include: paclitaxel, docetaxel, and related analogs; vincristine, vinblastin, and related analogs; thalidomide, lenalidomide, and related analogs (e.g., CC-5013 and CC-4047); protein tyrosine kinase inhibitors (e.g., imatinib mesylate and gefitinib); proteasome inhibitors (e.g., bortezomib, CEP-18770, MG132, peptide vinyl sulfones, peptide epoxyketones (such as epoxomicin and

carfilzomib), beta-lactone inhibitors (such as lactacystin, MLN 519, NPI-0052,

Salinosporamide A), compounds that create dithiocarbamate complexes with metals (such as Disulfiram), and certain antioxidants (such as Epigallocatechin-3-gallate, catechin-3- gallate, and Salinosporamide A); NF-kB inhibitors, including inhibitors of IKB kinase;

antibodies which bind to proteins overexpressed in cancers and thereby downregulate cell replication (e.g., trastuzumab, rituximab, cetuximab, and bevacizumab); and other inhibitors of proteins or enzymes known to be upregulated, over-expressed or activated in cancers, the inhibition of which downregulates cell replication.

[0093] The therapeutic regimen may comprise administration of other antibody

therapeutics, such as elotuzumab (a humanized monoclonal against SLAMF7; Tai et al., Blood, 2008;112:1329-37); daratumumab, MOR202, and isatuximab that target CD38;

nBT062-SMCC-DMl, nBT062-SPDB-DM4, and nBT062-SPP-DMl that target CD138;

lucatumumab (also known as HCD122) and dacetuzumab (also known as SGN-40) that target CD40; Lorvotuzumab which targets CD56. For a review of antibody therapeutics for the treatment of multiple myeloma, see, e.g., Tandon et al., Oncology & Hematology Review, 2015;11(2):115-21, and Sondergeld et al., Clinical Advances in Hematology & Oncology, 2015; 13(9), 599, both incorporated by reference.

[0094] In some embodiments, the heterodimeric antibody is administered prior to, concurrent with, or after treatment with Velcade® (bortezomib), Thalomid™ (thalidomide), Aredia™ (pamidronate), or Zometa™ (zoledronic acid). [0095] All cited references are herein expressly incorporated by reference in their entirety. Whereas particular embodiments of the invention have been described above for purposes of illustration, it will be appreciated by those skilled in the art that numerous variations of the details may be made without departing from the invention as described in the appended claims.

EXAMPLE

[0096] This Example is provided to further illustrate aspects of the method of the disclosure. The Example is not meant to constrain the invention to any particular application or theory of operation.

Preclinical studies

[0097] The heterodimeric antibody comprising a first monomer comprising the amino acid sequence of SEQ ID NO: 335, a second monomer comprising the amino acid sequence of SEQ ID NO: 82, and a light chain comprising the amino acid sequence of SEQ ID NO: 84 (referenced below as "Ab-A") is a highly specific and potent molecule able to induce human T cells to kill a variety of CD38-positive tumor cell lines with half-maximal effective concentrations (EC50) in the range of 0.65 ± 0.3 ng/mL to 21.77 ± 5.22 ng/mL (5.2 pM to 174.2 pM). The potency of Ab-A with cynomolgus monkey T cells against human cancer cells or cynomolgus monkey B cells was in the range of 2.17 ± 0.4 ng/mL to 12.33 ± 0.68 ng/mL (17.6 pM to 98.46 pM). EC50 values for cytotoxicity against CD38-positive cells varied between tumor cell lines with a difference between the most sensitive and least sensitive cell lines of 33.5-fold. However, this comparison is based on assays conducted under different conditions, including different effector cells (peripheral blood mononuclear cells (PBMCs) vs. purified T cells), different read-outs (Flow based vs. luciferase) and cell lines from a variety of indications (Multiple Myeloma, Acute Myeloid Leukemia, B cell lymphoma, Histiocytic Lymphoma). When assay conditions were standardized across four multiple myeloma cell lines with varying CD38 surface expression levels, the difference between the most sensitive and least sensitive multiple myeloma cell line was a much narrower 4-fold.

[0098] Ab-A efficiently engaged and activated human T cells in the presence of CD38 positive target cells resulting in target lysis accompanied by increased expression of T cell markers CD69, CD25 and CD38, increased T cell size, T cell expansion and release of pro- inflammatory cytokines IFN-g and TNF-oc. A similar activation of T cells was observed with cynomolgus monkey T cells or in autologous B cell depletion assays with cynomolgus monkey PBMCs and data derived from the in vitro studies demonstrate that the cynomolgus monkey is a pharmacologically relevant animal species for toxicity testing. However, cynomolgus monkeys T cells express significantly higher levels of CD38 than human T cells. While T cell expansion was observed in human T cell redirected lysis assays, T cell numbers decreased or stayed constant during the course of similar assays using cynomolgus monkey PBMCs, suggesting a differential effect of Ab-A on T cells between the two species.

[0099] Soluble CD38 is present in both species at very different concentrations: in multiple myeloma patients, the mean sCD38 concentration is 0.39 ng/mL (compared to 0.085 ng/mL in healthy individuals) with a maximal detected value of 2.82 ng/mL (n = 44), whereas in cynomolgus monkeys, serum sCD38 concentrations ranged from 0 to 247.8 ng/mL. In vitro, Ab-A-induced redirected lysis was unaffected by a 2 ng/mL concentration of soluble human CD38. Soluble cynomolgus monkey CD38 at a concentration of 200 ng/mL induced a modest 2-fold increase in the redirected lysis EC50 of Ab-A. Thus, sCD38 levels in multiple myeloma patients are unlikely to interfere with Ab-A activity. In addition, sCD38 at a 200 ng/mL concentration had no impact on T cell activation in the presence of Ab-A without target cells, demonstrating that the sCD38 concentrations commonly observed in patients are insufficient to trigger Ab-A -mediated T cell activation.

[00100] Ab-A induces the production of pro-inflammatory cytokines by T cells, including TNF-oc and IFN-g, in the presence of CD38-positive target cells. The effect of dexamethasone on Ab-A activity was assessed. Human T cells were co cultured with KMS- 12-BM luc MM target cells at an E:T ratio of 1 in the presence of dexamethasone and increasing concentrations of Ab-A. Effector cells and target cells were pre-treated with gradually decreasing concentrations of dexamethasone, starting at 230 ng/mL and ending at 12.6 ng/mL after 24 hours, corresponding to the serum concentration in human subjects 24 hours after a 20 mg oral dexamethasone dose. A redirected lysis assay was then performed with Ab-A at an E:T ratio of 1, at a continuous concentration of dexamethasone of 12.6 ng/mL. Under these clinically relevant conditions, the killing curves of KMS-12-BM luc MM target cells in the presence or absence of dexamethasone look very similar. The Ab-A target lysis EC50 was increased less than 2-fold at an E:T ratio of 1, but IFN-g and TNF-oc cytokine levels were reduced by more than 85%. See Figure 21. The data suggest that Ab-A could be administered 24 hours after oral administration of dexamethasone with limited impact on the potency of Ab-A, but with a possible benefit of lower cytokine release.

[00101] The in vivo efficacy of Ab-A is supported by data showing its anti-tumor effect in the MOLM-13 luc orthotopic mouse xenograft model supplemented with human T cells. Treatment with Ab-A significantly prolonged survival of mice bearing established orthotopic MOLM-13 luc tumors. NOD Scid gamma (NSG) immuno-compromised mice were orthotopically transplanted with CD38-expressing MOLM-13 luc tumor cells and intraperitoneally injected with human T cells 2 days later. Ab-A was administered by IV bolus injection at 0.01, 0.1, or 1 mg/kg from day 5 onwards every 7 days for 35 days.

Survival data are presented graphically in a Kaplan-Meier plot. Ab-A administration resulted in prolonged median survival (37.5, 36, and 37 days at the 0.01, 0.1, and 1 mg/kg dose levels, respectively) compared with the vehicle treated control group (22.0 days). Intergroup comparison of the survival using the log-rank test regarding vehicle as control demonstrated that Ab-A -induced prolongation of survival was highly statistically significant (p < 0.0001). A lack of a dose response was noted, as the maximal effect was observed at the lowest dose of 0.01 mg/kg. Tumor growth was monitored using whole body bioluminescence (BLI) imaging. One week after administration of the first dose on day 4, representative BLI images showed obvious elimination of the tumor in the hind limb and spine areas in all treatment groups compared with vehicle control animals. By day 22, tumor growth inhibition (TGI) at the 0.01, 0.1, and 1.0 mg/kg Ab-A dose levels was 98%,

93%, and 99%, respectively, and no overt body weight loss was observed.

[00102] In addition, Ab-A was shown to be pharmacologically active in cynomolgus monkeys in vivo, as indicated by Ab-A-induced depletion of peripheral B cells, activation of T cells and depletion of peripheral T cells. Ab-A triggers B cell depletion in vitro in an autologous redirected lysis assay using cynomolgus monkey PBMCs. To monitor the in vivo pharmacodynamics of Ab-A, the fate of peripheral B cells was also evaluated in cynomolgus monkeys after IV bolus administration on days 2, 5, 8, and 11 at a dose of 150 pg/kg after a single dose of 10 pg/kg on day 1 (n = 3/group). A decrease in peripheral B cells was observed after the administration of the first dose, with levels returning toward baseline before administration of the dose on day 5. By day 11, a profound mean decrease of 95.5% in peripheral B cell numbers compared with the predose levels was observed. In 1 animal, B cell numbers recovered after day 11 and this recovery was accompanied by a decrease in Ab- A serum levels. In a second cynomolgus monkey study of Ab-A, a dose-dependent transient decrease in peripheral B cell numbers and T cell activation on day 4 were also observed, confirming that Ab-A is active in vivo in the cynomolgus monkey and triggers T cell activation and CD38-positive cell depletion, both anticipated consequences of the T cell recruiting mechanism of action of Ab-A. Ab-A did not affect neuro, respiratory, or cardiovascular safety pharmacology parameters in the cynomolgus monkey toxicology study. Expression analysis identified the hematopoietic/lymphoid cells and tissues as most strongly-expressing CD38, and the safety evaluation in cynomolgus monkeys is consistent with this expression pattern.

[00103] Efficacy and specificity of Ab-A were demonstrated in several in vitro and in vivo studies, and the cynomolgus monkey was validated as a relevant species for the evaluation of safety.

Clinical study

[00104] Subjects will be enrolled to the dose exploration cohorts to estimate the maximum tolerated dose (MTD), safety, tolerability, PK, and PD of different doses of Ab-A in subjects with relapsed or refractory multiple myeloma using a Bayesian logistic regression model (BLRM). Ab-A will initially be administered as IV infusion as follows: two times per week during weeks 1 and 2, one time per week during weeks 3 and 4, and one time every other week starting in week 5 and thereafter. An alternative dosing regimen is as follows: one time per week during weeks 1, 2, 3, and 4, and one time every other week starting in week 5 and thereafter. Planned dose levels (dose per infusion) for the dose exploration cohorts are as follows: 0.05 mg, 0.15 mg, 0.45 mg, 1.35 mg, 4 mg, 12 mg, 36 mg, 100 mg and 200 mg. Premedication with IV dexamethasone is performed within 1 hour prior to the start of Ab-A unless a contraindication for premedication exists; initial premedication will comprise 8 mg dexamethasone, with a decrease in dose to 4 mg if treatment is well tolerated.

[00105] Various parameters are measured during the course of treatment, including vital signs, liver chemistries, hematology parameters, renal function, and associated adverse events (AEs). Minimal residual disease (MRD) assessments may also be performed. MRD assessments may utilize, e.g., next-generation sequencing (NGS) of samples from bone marrow and/or blood and/or flow cytometry using bone marrow. Assessment may be performed pre-treatment (baseline), after one or more rounds of treatment, and/or after complete remission is identified following IMWG guidelines. Cytokine release syndrome (CRS), resulting from overproduction of cytokines associated with CD3 engagement, is associated with fever, rigors, fatigue, malaise, headache, mental status changes, dysphasia, tremors, dysmetria, gait abnormalities, seizure, dyspnea, tachypnea, hypoxemia,

tachycardia, hypotension, nausea, vomiting, transaminitis, hyperbilirubinemia, bleeding, hypofibrinogenemia, elevated D-dimer, and/or rash. These symptoms will be monitored. CRS is graded as follows: Grade 1, symptoms are not life threatening and require symptomatic treatment only (e.g., fever, nausea, fatigue, headache, myalgias, malaise);

Grade 2, symptoms require and respond to moderate intervention (Oxygen requirement < 40%, OR Hypotension responsive to fluids or low dose of 1 vasopressor, OR Grade 2 organ toxicity or grade 3 transaminitis per CTCAE criteria); Grade 3, symptoms require and respond to aggressive intervention (Oxygen requirement > 40%, OR Hypotension requiring high dose or multiple vasopressors, OR Grade 3 organ toxicity or grade 4 transaminitis per CTCAE criteria); and Grade 4, life-threatening symptoms (requirement for ventilator support OR Grade 4 organ toxicity (excluding transaminitis) per CTCAE criteria). In various aspects, the subject experiences little or no CRS symptoms (e.g., the subject experiences Grade 2, Grade 1, or no CRS.

[00106] Methods of determining overall response rate (ORR) per International

Myeloma Working Group (IMWG) response criteria (stringent [s] Complete Response [CR], CR, very good [VG] Partial Response [PR], PR), duration of response, time to progression, progression-free survival, and overall survival are known in the art. Complete response (CR) is characterized by, e.g., negative immunofixation on the serum and urine and disappearance of any soft tissue plasmacytomas and < 5% plasma cells in bone marrow. Stringent complete response is characterized by CR as defined above alongside normal FLC ratio and absence of clonal cells in bone marrow by immunohistochemistry or

immunofluorescence. VGPR is characterized by, e.g., serum and urine M-protein detectable by immunofixation but not on electrophoresis or > 90% reduction in serum M-protein plus urine M-protein level < 100 mg/24 h. PR is characterized by, e.g., > 50% reduction of serum M-protein and reduction in 24 hours urinary M-protein by >90% or to < 200 mg/24 h; if the serum and urine M-protein are unmeasurable, a > 50% decrease in the difference between involved and uninvolved FLC levels is required in place of the M-protein criteria; if serum and urine M-protein are not measurable, and serum free light assay is also not measureable, > 50% reduction in plasma cells is required in place of M-protein, provided baseline bone marrow plasma cell percentage was > 30%; and if present at baseline, a > 50% reduction in the size of soft tissue plasmacytomas. See, e.g., the International Myeloma Working Group (IMWG) Uniform Response Criteria for Multiple Myeloma available at:

imwg.myeloma.org/international-myeloma-working-group-imwg-uniform-response- criteria-for-multiple-myeloma/. The disclosure contemplates improvement of any of these parameters, and preferable improvement sufficient to achieve at least PR, at least VGPR,CR, or sCR.

Claims

WE CLAIM:

1. A method of treating multiple myeloma, the method comprising administering to a subject in need thereof a heterodimeric antibody comprising

a) a first monomer comprising a first Fc domain and an anti-CD3 scFv comprising

(i) a scFv variable light domain comprising vlCDRl as set forth in SEQ ID NO:15, vlCDR2 as set forth in SEQ ID NO:16, and vlCDRB as set forth in SEQ ID NO: 17, and

(ii) a scFv variable heavy domain comprising vhCDRl as set forth in SEQ ID NO:ll, vhCDR2 as set forth in SEQ ID NO:12, and vhCDR3 as set forth in SEQ ID NO:13, wherein said scFv is covalently attached to the N-terminus of said Fc domain using a domain linker;

b) a second monomer comprising

i) an anti-CD38 heavy variable domain comprising vhCDRl as set forth in SEQ ID NO:65, vhCDR2 as set forth in SEQ ID NO:66, and vhCDR3 as set forth in SEQ ID NO:67, and

ii) a heavy constant domain comprising a second Fc domain and; and c) a light chain comprising a variable constant domain and an anti-CD38 variable light domain comprising vlCDRl as set forth in SEQ ID NO:69, vlCDR2 as set forth in SEQ ID NO: 70, and vlCDR3 as set forth in SEQ ID NO: 71,

in a dose of about 0.05 mg to about 200 mg.

2. The method of claim 1, wherein the dose is about 0.05 mg, 0.15 mg, 0.45 mg, 1.35 mg, 4 mg, 12 mg, 36 mg, 100 mg, or 200 mg.

3. The method of claim 1, wherein the dose is about 36 mg to about 200 mg.

4. The method of any one of claims 1-3, comprising administering two doses of heterodimeric antibody per week to the subject in the first and second weeks of treatment, administering one dose of heterodimeric antibody per week to the subject in the third and fourth weeks of treatment, and administering one dose of heterodimeric antibody every two weeks starting in week 5 through the end of treatment.

5. The method of any one of claims 1-3, comprising administering one dose of heterodimeric antibody per week to the subject in the first, second, third, and fourth weeks of treatment, and administering one dose of heterodimeric antibody every two weeks starting in week 5 through the end of treatment.

6. The method of any one of claims 1-5, wherein the method comprises administering two or more doses of the heterodimeric antibody over a treatment period of about six months to about 12 months.

7. The method of claim 6, wherein the treatment period is about eight months.

8. The method of any one of claims 1-7, wherein the heterodimeric antibody is administered via intravenous infusion over a period of about 30 minutes to about four hours.

9. The method of claim 8, wherein a first dose of heterodimeric antibody is

administered over a period of about four hours, a second dose of heterodimeric antibody is administered over a period of about two hours, and subsequent doses are administered over a period of about 30 minutes.

10. The method of any one of claims 1-9, comprising administering two or more doses of heterodimeric antibody and increasing the dose at least once during treatment.

11. The method of any one of claims 1-10, wherein the subject is suffering from relapsed/refractory multiple myeloma.

12. The method of any one of claims 1-11, further comprising administering

dexamethasone to the subject.

13 The method of claim 12, wherein the dexamethasone is administered intravenously.

14. The method of claim 12 or claim 13, wherein the dexamethasone is administered to the subject within one hour prior to administration of the heterodimeric antibody.

15. The method of any one of claims 12-14, wherein the dexamethasone is administered in an amount of about 8 mg or about 4 mg.

16. The method of claim 12, wherein the dexamethasone is administered orally.

17. The method of any one of claims 1-16, comprising (a) administering to the subject an anti-CD38 monospecific antibody and, following a wash-out period sufficient to reduce systemic concentration of the anti-CD38 monospecific antibody to 0.2 pg/ml or less, (b) administering an initial dose of the heterodimeric antibody.

18. The method of any one of claims 1-16, comprising (a) administering to the subject an anti-CD38 monospecific antibody and (b) administering an initial dose of the heterodimeric antibody at a timepoint that is at least 12 weeks after administration of the anti-CD38 monospecific antibody has ceased.

19. The method of claim 18, wherein the method comprises ceasing treatment of the anti- CD38 monospecific antibody 14-16 weeks prior to administering an initial dose of the heterodimeric antibody.

20. The method of any one of claims 17-19, wherein the anti-CD38 monospecific antibody is daratumumab.

21. The method of any one of claims 1-20, wherein the subject was previously treated with a proteasome inhibitor and/or an immunomodulatory drug.

22. The method of any one of claims 1-21, wherein the anti-CD3 scFv comprises an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 18.

23. The method of any one of claims 1-21, wherein the anti-CD3 scFv comprises the amino acid sequence set forth in SEQ ID NO: 18.

24. The method of any one of claims 1-23, the anti-CD38 variable light domain comprises an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO:68.

25. The method of claim 24, the anti-CD38 variable light domain comprises the amino acid sequence set forth in SEQ ID NO:68.

26. The method of any one of claims 1-25, wherein the anti-CD38 heavy variable domain comprises an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO:64.

27. The method of claim 26, wherein the anti-CD38 heavy variable domain comprises the amino acid sequence set forth in SEQ ID NO:64.

28. The method of any one of claims 1-27, wherein the first monomer comprises an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO:335.

29. The method of claim 28, wherein the first monomer comprises the amino acid sequence set forth in SEQ ID NO:335.

30. The method of any one of claims 1-29, wherein the second monomer comprises an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO:82.

31. The method claim 30, wherein the second monomer comprises the amino acid sequence set forth in SEQ ID NO:82.

32. The method of any one of claims 1-31, wherein the light chain comprises an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO:84.

33. The method of claim 32, wherein the light chain comprises the amino acid sequence set forth in SEQ ID NO:84.

34. The method of any one of claims 1-27, wherein the first Fc domain and the second Fc domain comprises one or more mutations that reduce homodimerization.

35. The method of any one of claims 1-27, wherein said first Fc domain and said second Fc domain comprise a set of variants selected from the group consisting of S364K/E357Q : F368D/K370S; F368D/K370S : S364K; F368E/K370S : S364K; T411T/E360E/Q362E : D401K; F368D/K370S : S364K/E357F and K370S : S364K/E357Q.

36. The method of any one of claims 1-27, wherein said scFv domain linker is a charged linker.

37. The method of any one of claims 1-27, wherein said heavy chain constant domain comprises the amino acid substitutions N208D/Q295E/N384D/Q418E/N421D.

38. The method of any one of claims 1-27, wherein said first and second Fc domains comprise the amino acid substitutions E233P/F234V/F235A/G236del/S267K.