Antigen-binding molecules that bind CD38 and / or CD28 and their use

Abstract

CD38 is expressed on malignant plasma cells. CD28 is a costimulatory molecule required for T cell activation and survival. Provided herein are novel anti-CD38 antibodies, anti-CD28 antibodies, and bispecific antibodies (bsAbs) that bind to both CD38 and CD28 and act as costimulators to activate T cells through binding to CD80 and / or CD86. In certain embodiments, the bispecific antigen-binding molecules of the present invention can inhibit the growth of CD38-expressing tumors. The bispecific antigen-binding molecules of the present invention are useful for treating diseases and disorders in which an upregulated or induced CD38-targeted immune response is desirable and / or therapeutically beneficial. For example, the bispecific antibodies of the present invention are useful for treating various cancers, including multiple myeloma, lymphoma, and leukemia.

Description

[Technical Field]

[0001] The present invention relates to antigen-binding molecules comprising antibodies and antigen-binding fragments thereof, as well as bispecific antigen-binding molecules specific to CD38 and / or CD28 (e.g., bispecific antibodies), and methods for using them.

[0002] Sequence List A formal copy of the sequence listing will be submitted electronically via EFS-Web at the same time as this Specified, as an ASCII format sequence listing with the filename "10786WO01_Sequence_Listing_ST25", created on September 17, 2021, and approximately 56KB in size. The sequence listing contained in this ASCII format document is part of this Specified, and the entirety is incorporated herein by reference. [Background technology]

[0003] Multiple myeloma (MM) is the second most common blood cancer after non-Hodgkin lymphoma, with a prevalence of approximately 120,000 people in the United States, and approximately 30,000 new cases and 13,000 deaths annually. MM is characterized by the clonal proliferation of malignant plasma cells that secrete cytokines in an uncontrolled manner. The production of cytokines, particularly IL-6, causes local organ and tissue damage that leads to many of the symptoms associated with myeloma. MM patients suffer from bone pain and osteoporosis, anemia, renal dysfunction and failure, bacterial infections, and neurological disorders. MM has an average life expectancy of 4-5 years and is rarely curable. While treatment for MM has advanced, new therapies have been providing disproportionate benefits to younger patients. The prognosis for patients with relapsed MM is poor, and new therapeutic approaches are urgently needed.

[0004] CD38, also known as cyclic ADP-ribose hydrolase, is a 45 kDa surface glycoprotein expressed on thymocytes, some activated peripheral blood T cells and B cells, plasma cells, and dendritic cells. CD38 is an extracellular nicotinamide adenine dinucleotide (NAD +It functions as an exogenous enzyme involved in the metabolism of cyclic adenosine diphosphate (ADP) ribose, ADP-ribose (ADPR), and nicotinic acid adenine dinucleotide phosphate (NADP) (Howard, et al. Formation and hydrolysis of cyclic ADP-ribose catalyzed by lymphocyte antigen CD38. Science (1993) 262:1056-9), and Ca2+ ribose, ADP-ribose (ADPR), and nicotinic acid adenine dinucleotide phosphate. 2+ It leads to the production of mobilization compounds. Calcium regulation results in the activation of signaling pathways that control a wide range of physiological functions, including lymphocyte proliferation, insulin release by the pancreas, myocardial contraction, neutrophil chemotaxis, and T cell activation. CD38 enzyme activity is linked to NAD + It modulates levels and improves the function of proteasome inhibitors (Cagnetta, et al. Intracellular NAD(+) depletion enhances bortezomib-induced anti-myeloma activity. Blood (2013) 122:1243-55). Furthermore, ADPR can be metabolized by CD203a / PC-1 and CD73 to produce the immunosuppressive molecule adenosine (ADO), which can promote the escape of tumor cells from immune system control (Chillemi et al. Roles and modalities of ectonucleotidases in remodeling the multiple myeloma niche. Front Immunol. (2017) 8:305). CD38 appears to contribute to the proliferative capacity of chronic leukemia B / small lymphocytic lymphoma, and malignant plasma cells in the bone marrow express high and uniform levels of CD38. Anti-CD38 mAbs are thought to deplete CD38+ immunosuppressive cells, such as myeloid-derived suppressor cells, regulatory T cells, and regulatory B cells, leading to increased antitumor activity of immune effector cells. Daratumumab, an anti-CD38 antibody, is approved for patients with multiple myeloma who are resistant to conventional treatments.

[0005] T cell activation involves highly specific stimulation of the T cell receptor (TCR) by antigen-presenting cells (such as dendritic cells) that present their specific antigen on their class II major histocompatibility complex (MHC), and can be promoted by costimulatory molecules such as CD28. CD28 is a 44 kDa disulfide-linked homodimeric receptor that is glycosylated at five different sites. CD28 is expressed on T cells (95% of quiescent CD4+ cells and 50% of quiescent CD8+ T cells in human peripheral blood) and plasmablasts, and provides costimulatory signaling necessary for T cell activation and survival. Costimulation occurs when CD28 on the surface of T cells binds to CD80 (B7-1) and CD86 (B7-2) on antigen-presenting cells. CD80 expression is upregulated in antigen-presenting cells (APCs) when activated, and CD86 is constitutively expressed on APCs. CD28 co-stimulation of helper T cells enhances the transcription of IL-2R and IL-2 (leading to T cell proliferation), induces the expression of Bcl-XL (enhancing T cell survival), increases the production of IL-4 (leading to Th2 differentiation), IFNγ, IL-1, TNF, IL-5, various chemokines, and their receptors. Furthermore, CD28 induces the expression or upregulation of several other costimulatory and regulatory molecules, including ICOS, 4-1BB, and CTLA-4, along with the CD40L molecule necessary for TB cell interaction. (Mak and Saunders, The Immune Response, Basic and Clinical Principles, Academic Press, 2006). TGN1412 regulates T(T) cells in the absence of TCR co-stimulation. Reg It is a CD28 superagonist monoclonal antibody that preferentially activates ) cells. Attarwala, TGN1412: From Discovery to Disaster. J Young Pharm. 2010, 2(3):332-6. Unfortunately, in a Phase I clinical trial, all six volunteers developed rapid multiple organ failure and severe cytokine release syndrome (cytokine storm).

[0006] Therefore, there is a need in this technology field for alternative approaches to treating cancer. [Overview of the project]

[0007] The present invention relates in part to monospecific antibodies that bind to CD38 or CD28, as well as bispecific antibodies that bind to both CD38 and CD28, and to their use in the treatment of various diseases, including cancer.

[0008] Antibodies or bispecific antibodies can be used alone or in combination with other agents for treating cancers that express CD38.

[0009] Anti-CD38 antibody and its antigen-binding fragment In one embodiment, antibodies that bind to human CD38 and antigen-binding fragments thereof are provided herein. The antibodies may be particularly useful for targeting cells expressing CD38 and / or inducing apoptosis. In certain embodiments, the antibodies may be useful for mediating antibody-dependent cell-mediated cytotoxicity (ADCC) and / or complement-mediated cytotoxicity (CDC) against CD38+ cancer cells. The anti-CD38 antibodies or antigen-binding moieties provided herein may be included as part of a bispecific antibody that promotes CD38-targeted cell-mediated cytotoxicity of specific cell types, such as tumor cells.

[0010] Exemplary anti-CD38 antibodies provided herein are listed in Tables 1 and 2. Table 1 lists the amino acid sequence identifiers for the heavy chain variable region (HCVR) and light chain variable region (LCVR), as well as the heavy chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) and light chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3) of the exemplary anti-CD38 antibodies. Table 2 lists the sequence identifiers for the nucleic acid molecules encoding the HCVR, LCVR, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of the exemplary anti-CD38 antibodies.

[0011] This specification provides an anti-CD38 antibody or an antigen-binding fragment thereof, comprising an HCVR containing an amino acid sequence selected from any of the HCVR amino acid sequences listed in Table 1, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0012] This specification also provides anti-CD38 antibodies or antigen-binding fragments thereof, comprising an LCVR containing an amino acid sequence selected from any of the LCVR amino acid sequences listed in Table 1, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0013] This specification provides an anti-CD38 antibody or an antigen-binding fragment thereof comprising an HCVR / LCVR amino acid sequence pair (HCVR / LCVR) containing one of the HCVR amino acid sequences listed in Table 1, which is paired with any of the LCVR amino acid sequences listed in Table 1. According to a particular embodiment, the present invention provides an antibody or an antigen-binding fragment thereof comprising an HCVR / LCVR amino acid sequence pair contained within one of the exemplary anti-CD38 antibodies listed in Table 1. In a particular embodiment, the HCVR / LCVR amino acid sequence pair is selected from the group consisting of SEQ ID NOs: 2 / 18 (e.g., mAb1) and 32 / 48 (e.g., mAb2).

[0014] This specification provides an anti-CD38 antibody or an antigen-binding fragment thereof comprising a heavy chain CDR1 (HCDR1) containing an amino acid sequence selected from any of the HCDR1 amino acid sequences listed in Table 1, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0015] This specification provides an anti-CD38 antibody or an antigen-binding fragment thereof comprising a heavy chain CDR2 (HCDR2) containing an amino acid sequence selected from any of the HCDR2 amino acid sequences listed in Table 1, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0016] This specification provides an anti-CD38 antibody or an antigen-binding fragment thereof, comprising a heavy chain CDR3 (HCDR3) containing an amino acid sequence selected from any of the HCDR3 amino acid sequences listed in Table 1, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0017] This specification provides an anti-CD38 antibody or an antigen-binding fragment thereof, comprising a light chain CDR1 (LCDR1) containing an amino acid sequence selected from any of the LCDR1 amino acid sequences listed in Table 1, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0018] This specification provides an anti-CD38 antibody or an antigen-binding fragment thereof, comprising a light chain CDR2 (LCDR2) containing an amino acid sequence selected from any of the LCDR2 amino acid sequences listed in Table 1, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0019] This specification provides an anti-CD38 antibody or an antigen-binding fragment thereof, comprising a light chain CDR3 (LCDR3) containing an amino acid sequence selected from any of the LCDR3 amino acid sequences listed in Table 1, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0020] This specification provides an anti-CD38 antibody or an antigen-binding fragment thereof comprising an HCDR3 / LCDR3 amino acid sequence pair (HCDR3 / LCDR3) containing one of the HCDR3 amino acid sequences listed in Table 1, which is paired with any of the LCDR3 amino acid sequences listed in Table 1. According to one embodiment, the antibody or the antigen-binding fragment thereof comprises an HCDR3 / LCDR3 amino acid sequence pair contained within one of the exemplary anti-CD38 antibodies listed in Table 1. In a particular embodiment, the HCDR3 / LCDR3 amino acid sequence pair is selected from the group consisting of SEQ ID NOs: 8 / 24 (e.g., mAb1) and 38 / 54 (e.g., mAb2).

[0021] This specification provides antibodies or antigen-binding fragments thereof containing a set of six CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained within any of the exemplary anti-CD38 antibodies listed in Table 1. In certain embodiments, the HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequence set is selected from the group consisting of SEQ ID NOs: 4-6-8-20-22-24 (e.g., mAb1) and SEQ ID NOs: 34-36-38-50-52-54 (e.g., mAb2).

[0022] In related embodiments, this specification provides an antibody or an antigen-binding fragment thereof comprising a set of six CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained within an HCVR / LCVR amino acid sequence pair defined by one of the exemplary anti-CD38 antibodies listed in Table 1. For example, this specification provides an antibody or an antigen-binding fragment thereof comprising the HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequence set contained within an HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 2 / 18 (e.g., mAb1) and 32 / 48 (e.g., mAb2).

[0023] In the embodiments provided herein, the anti-CD38 antibody or its antigen-binding fragment is HCDR1 containing the amino acid sequence: GFTFDDYA (SEQ ID NO: 4, or its variant), HCDR2 containing the amino acid sequence: ISWKSDNI (SEQ ID NO: 6, or its variant), and HCDR3 containing the amino acid sequence: AKALGGWKFDYYYGMDV (SEQ ID NO: 8, or its variant), and LCDR1 contains the amino acid sequence QSISSY (SEQ ID NO: 20 or its variant), LCDR2 contains the amino acid sequence AAS (SEQ ID NO: 22 or its variant), and LCDR3 contains the amino acid sequence QQSYSTPPIT (SEQ ID NO: 24 or its variant).

[0024] In the embodiments provided herein, the anti-CD38 antibody or its antigen-binding fragment is HCDR1 containing amino acid sequence: GGPFRSSS (SEQ ID NO: 34, or its variant), HCDR2 containing amino acid sequence: IIPILGKT (SEQ ID NO: 36, or its variant), and HCDR3 containing amino acid sequence: VRGSSLFDY (SEQ ID NO: 38, or its variant), LCDR1 contains the amino acid sequence QSVSSSY (SEQ ID NO: 50 or its variant), LCDR2 contains the amino acid sequence GAS (SEQ ID NO: 52 or its variant), and LCDR3 contains the amino acid sequence QQYGSSPWT (SEQ ID NO: 54 or its variant).

[0025] In the embodiments provided herein, the anti-CD38 antibody or its antigen-binding fragment is HCVR1 containing the amino acid sequence:EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGISWKSDNIGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCAKALGGWKFDYYYGMDVWGQGTTVTVSS (SEQ ID NO: 2, or its variant), The amino acid sequence is DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPITFGQGTRLEIK (Sequence ID 18, or a variant thereof), and the LCVR1 comprises this sequence.

[0026] In the embodiments provided herein, the anti-CD38 antibody or its antigen-binding fragment is HCVR1 containing the amino acid sequence: QVQLVQSGAEVKKPGSSVKVSCKASGGPFRSSSFSWVRQAPGQGLEWMGGIIPILGKTNYAQKFQGRITIVTDESTTTVYMELSSLRSEDTAVFYCVRGSSLFDYWGQGTLVTVSS (SEQ ID NO: 32, or its variant), The amino acid sequence is EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIK (SEQ ID NO: 48, or a variant thereof), and LCVR1 is included.

[0027] Nucleic acid molecules encoding anti-CD38 antibodies or portions thereof are also provided herein. For example, the present invention provides a nucleic acid molecule encoding any of the HCVR amino acid sequences listed in Table 1, and in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCVR nucleic acid sequences listed in Table 2, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity therewith.

[0028] Nucleic acid molecules encoding any of the LCVR amino acid sequences listed in Table 1 are provided herein, and in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCVR nucleic acid sequences listed in Table 2, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity therewith.

[0029] Nucleic acid molecules encoding any of the HCDR1 amino acid sequences listed in Table 1 are provided herein, and in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCDR1 nucleic acid sequences listed in Table 2, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity therewith.

[0030] Nucleic acid molecules encoding any of the HCDR2 amino acid sequences listed in Table 1 are provided herein, and in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCDR2 nucleic acid sequences listed in Table 2, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity therewith.

[0031] Nucleic acid molecules encoding any of the HCDR3 amino acid sequences listed in Table 1 are provided herein, and in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCDR3 nucleic acid sequences listed in Table 2, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity therewith.

[0032] Nucleic acid molecules encoding any of the LCDR1 amino acid sequences listed in Table 1 are provided herein, and in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCDR1 nucleic acid sequences listed in Table 2, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity therewith.

[0033] Nucleic acid molecules encoding any of the LCDR2 amino acid sequences listed in Table 1 are provided herein, and in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCDR2 nucleic acid sequences listed in Table 2, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity therewith.

[0034] Nucleic acid molecules encoding any of the LCDR3 amino acid sequences listed in Table 1 are provided herein, and in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCDR3 nucleic acid sequences listed in Table 2, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity therewith.

[0035] Nucleic acid molecules encoding HCVR are provided herein, HCVR comprising a set of three CDRs (i.e., HCDR1-HCDR2-HCDR3), the set of HCDR1-HCDR2-HCDR3 amino acid sequences as defined by any of the exemplary anti-CD38 antibodies listed in Table 1.

[0036] A nucleic acid molecule encoding LCVR is provided herein, which comprises a set of three CDRs (i.e., LCDR1-LCDR2-LCDR3), the set of amino acid sequences of LCDR1-LCDR2-LCDR3 as defined by one of the exemplary anti-CD38 antibodies listed in Table 1.

[0037] Nucleic acid molecules encoding both HCVR and LCVR are provided herein, where HCVR comprises an amino acid sequence from any of the HCVR amino acid sequences listed in Table 1, and LCVR comprises an amino acid sequence from any of the LCVR amino acid sequences listed in Table 1. In certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCVR nucleic acid sequences listed in Table 2, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto, and a polynucleotide sequence selected from any of the LCVR nucleic acid sequences listed in Table 2, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto. In certain embodiments of this aspect of the present invention, the nucleic acid molecule encodes both HCVR and LCVR, and both HCVR and LCVR are derived from the same anti-CD38 antibody listed in Table 1.

[0038] This specification provides recombinant expression vectors capable of expressing polypeptides comprising the heavy chain variable region or light chain variable region of an anti-CD38 antibody. For example, this specification provides recombinant expression vectors comprising any of the nucleic acid molecules described above, namely, nucleic acid molecules encoding any of the HCVR sequence, LCVR sequence, and / or CDR sequence listed in Table 1. Host cells into which such vectors have been introduced, as well as methods for producing antibodies or portions thereof by culturing host cells under conditions that enable the production of antibodies or antibody fragments, and methods for recovering the antibodies and antibody fragments thus produced, are also included within the scope of this disclosure.

[0039] Anti-CD38 antibodies having modified glycosylation patterns are provided herein. In some embodiments, modifications to remove undesirable glycosylation sites, i.e., antibodies lacking the fucose portion present on the oligosaccharide chain, may be useful to enhance antitumor activity, such as antibody-dependent cell-mediated cytotoxicity (ADCC) function (see Shield et al. JBC 277:26733 2002). In other applications, galactosylation modifications can be made to modify complement-dependent cell-mediated cytotoxicity (CDC) activity.

[0040] In another embodiment, this specification provides a pharmaceutical composition comprising a recombinant human antibody or fragment thereof that specifically binds to CD38 and a pharmaceutically acceptable carrier. In a related embodiment, the present invention features a composition that is a combination of an anti-CD38 antibody and a second therapeutic agent. In one embodiment, the second therapeutic agent is any agent that is advantageously combined with the anti-CD38 antibody. Exemplary agents that can be advantageously combined with the anti-CD38 antibody include, but are not limited to, agents that bind to and / or inactivate CD38 signaling (including other antibodies or their antigen-binding fragments) and / or agents that do not directly bind to CD38 but activate or stimulate immune cells. Additional combination therapies and combinations involving the anti-CD38 antibody of the present invention are disclosed elsewhere in this specification.

[0041] In yet another embodiment, a therapeutic method for promoting the treatment of cancer using an anti-CD38 antibody or an antigen-binding portion of an anti-CD38 antibody disclosed herein is provided herein, the therapeutic method comprising administering a therapeutically effective amount of a pharmaceutical composition comprising an antibody or antigen-binding fragment of an anti-CD38 antibody to a subject in need thereof. The disorder to be treated is any disease or condition that is improved, remissioned, inhibited, or prevented by inhibition of CD38 activity or signaling.

[0042] Anti-CD28 antibody and its antigen-binding fragment In another embodiment, antibodies that bind to human CD28 and antigen-binding fragments thereof are provided herein. Antibodies according to this embodiment of the present invention are particularly useful for binding to CD28-expressing immune cells, including CD4+, CD8+, plasma cells, and natural killer cells, and for co-stimulating T cell activation under circumstances where T cell-mediated killing is beneficial or desirable. The anti-CD28 antibody of the present invention, or its antigen-binding portion thereof, may be included as part of a bispecific antibody that directs T cell activation to a specific cell type, such as tumor cells.

[0043] Examples of anti-CD28 antibodies provided herein are listed in Tables 4 and 5. Table 4 lists the amino acid sequence identifiers of the heavy chain variable region (HCVR) and light chain variable region (LCVR), as well as the heavy chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) and light chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3) of the example anti-CD28 antibodies. Table 5 lists the sequence identifiers of the nucleic acid molecules encoding the HCVR, LCVR, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of the example anti-CD28 antibodies.

[0044] This specification provides an anti-CD28 antibody or an antigen-binding fragment thereof comprising an HCVR having an amino acid sequence selected from any of the HCVR amino acid sequences listed in Table 4, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity therewith.

[0045] This specification provides an anti-CD28 antibody or an antigen-binding fragment thereof comprising an LCVR containing an amino acid sequence selected from any of the LCVR amino acid sequences listed in Table 4, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity therewith.

[0046] This specification provides an anti-CD28 antibody or an antigen-binding fragment thereof comprising an HCVR-LCVR amino acid sequence pair (HCVR / LCVR) containing one of the HCVR amino acid sequences listed in Table 4, which is paired with any of the LCVR amino acid sequences listed in Table 4. According to a particular embodiment, this disclosure provides an antibody or an antigen-binding fragment thereof comprising an HCVR / LCVR amino acid sequence pair contained in one of the exemplary anti-CD28 antibodies listed in Table 4. In a particular embodiment, the HCVR / LCVR amino acid sequence pair is selected from the group consisting of SEQ ID NOs: 10 / 18 (e.g., mAb3) and 40 / 48 (e.g., mAb4).

[0047] This specification provides an anti-CD28 antibody or an antigen-binding fragment thereof comprising a heavy chain CDR1 (HCDR1) containing an amino acid sequence selected from any of the HCDR1 amino acid sequences listed in Table 4, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0048] This specification provides an anti-CD28 antibody or an antigen-binding fragment thereof, comprising a heavy chain CDR2 (HCDR2) containing an amino acid sequence selected from any of the HCDR2 amino acid sequences listed in Table 4, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0049] This specification provides an anti-CD28 antibody or an antigen-binding fragment thereof comprising a heavy chain CDR3 (HCDR3) containing an amino acid sequence selected from any of the HCDR3 amino acid sequences listed in Table 4, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0050] This specification provides an anti-CD28 antibody or an antigen-binding fragment thereof, comprising a light chain CDR1 (LCDR1) containing an amino acid sequence selected from any of the LCDR1 amino acid sequences listed in Table 4, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0051] This specification provides an anti-CD28 antibody or an antigen-binding fragment thereof, comprising a light chain CDR2 (LCDR2) containing an amino acid sequence selected from any of the LCDR2 amino acid sequences listed in Table 4, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0052] This specification provides an anti-CD28 antibody or an antigen-binding fragment thereof, comprising a light chain CDR3 (LCDR3) containing an amino acid sequence selected from any of the LCDR3 amino acid sequences listed in Table 4, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0053] This specification provides an anti-CD28 antibody or an antigen-binding fragment thereof comprising an HCDR3 / LCDR3 amino acid sequence pair (HCDR3 / LCDR3) containing one of the HCDR3 amino acid sequences listed in Table 4, which is paired with any of the LCDR3 amino acid sequences listed in Table 4. According to a particular embodiment, this disclosure provides an antibody or an antigen-binding fragment thereof comprising an HCDR3 / LCDR3 amino acid sequence pair contained in one of the exemplary anti-CD28 antibodies listed in Table 4. In a particular embodiment, the HCDR3 / LCDR3 amino acid sequence pair is selected from the group consisting of SEQ ID NOs: 16 / 24 (e.g., mAb3) and 46 / 54 (e.g., mAb4).

[0054] The present invention also provides an antibody or an antigen-binding fragment thereof containing a set of six CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained within any of the exemplary anti-CD28 antibodies listed in Table 4. In certain embodiments, the HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequence set is selected from the group consisting of SEQ ID NOs: 12-14-16-20-22-24 (e.g., mAb3) and SEQ ID NOs: 42-44-46-50-52-54 (e.g., mAb4).

[0055] In related embodiments, the disclosure provides an antibody or an antigen-binding fragment thereof comprising a set of six CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained within an HCVR / LCVR amino acid sequence pair defined by one of the exemplary anti-CD28 antibodies listed in Table 4. For example, the Specified provides an antibody or an antigen-binding fragment thereof comprising the HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequence set contained within an HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 10 / 18 (e.g., mAb3) and 40 / 48 (e.g., mAb4).

[0056] In the embodiments provided herein, the anti-CD28 antibody or its antigen-binding fragment is HCDR1 containing amino acid sequence: GFTFSRNN (SEQ ID NO: 12 or its variant), HCDR2 containing amino acid sequence: ISSNGGRT (SEQ ID NO: 14 or its variant), and HCDR3 containing amino acid sequence: TRDDELLSFDY (SEQ ID NO: 16 or its variant), and LCDR1 contains the amino acid sequence QSISSY (SEQ ID NO: 20 or its variant), LCDR2 contains the amino acid sequence AAS (SEQ ID NO: 22 or its variant), and LCDR3 contains the amino acid sequence QQSYSTPPIT (SEQ ID NO: 24 or its variant).

[0057] In the embodiments provided herein, the anti-CD28 antibody or its antigen-binding fragment is HCDR1 contains the amino acid sequence:GGSISSYY (SEQ ID NO: 42, or its variant), HCDR2 contains the amino acid sequence:IYYSGIT (SEQ ID NO: 44, or its variant), and HCDR3 contains the amino acid sequence:ARWGVRRDYYYYGMDV (SEQ ID NO: 46, or its variant), and LCDR1 contains the amino acid sequence QSVSSSY (SEQ ID NO: 50 or its variant), LCDR2 contains the amino acid sequence GAS (SEQ ID NO: 52 or its variant), and LCDR3 contains the amino acid sequence QQYGSSPWT (SEQ ID NO: 54 or its variant).

[0058] In the embodiments provided herein, the anti-CD28 antibody or its antigen-binding fragment is HCVR1 containing the amino acid sequence:EVQLVESGGGLVQPGGSLRLSCAASGFTFSRNNMHWVRQAPGKGLEYVSGISSNGGRTYYADSVKGRFTISRDNSKNTLYLQMGGLRAADMAVYFCTRDDELLSFDYWGQGTLVTVSS (SEQ ID NO: 10, or its variant), The amino acid sequence is DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPITFGQGTRLEIK (Sequence ID 18, or a variant thereof), and the LCVR1 comprises this sequence.

[0059] In the embodiments provided herein, the anti-CD28 antibody or its antigen-binding fragment is HCVR1 containing the amino acid sequence: QVQLQESGPGLVKPSETLSLTCTVSGGSISSYYWSWIRQPPGKGLEWIGYIYYSGITHYNPSLKSRVTISVDTSKIQFSLKLSSVTAADTAVYYCARWGVRRDYYYYGMDVWGQGTTVTVSS (SEQ ID NO: 40, or its variant), The amino acid sequence is EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIK (SEQ ID NO: 48, or a variant thereof), and LCVR1 is included.

[0060] Nucleic acid molecules encoding anti-CD28 antibodies or portions thereof are also provided herein. For example, the present invention provides a nucleic acid molecule encoding any of the HCVR amino acid sequences listed in Table 4, and in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCVR nucleic acid sequences listed in Table 5, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity therewith.

[0061] Nucleic acid molecules encoding any of the LCVR amino acid sequences listed in Table 4 are provided herein, and in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCVR nucleic acid sequences listed in Table 5, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity therewith.

[0062] Nucleic acid molecules encoding any of the HCDR1 amino acid sequences listed in Table 4 are provided herein, and in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCDR1 nucleic acid sequences listed in Table 5, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity therewith.

[0063] Nucleic acid molecules encoding any of the HCDR2 amino acid sequences listed in Table 4 are provided herein, and in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCDR2 nucleic acid sequences listed in Table 5, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity therewith.

[0064] Nucleic acid molecules encoding any of the HCDR3 amino acid sequences listed in Table 4 are provided herein, and in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCDR3 nucleic acid sequences listed in Table 5, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity therewith.

[0065] Nucleic acid molecules encoding any of the LCDR1 amino acid sequences listed in Table 4 are provided herein, and in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCDR1 nucleic acid sequences listed in Table 5, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity therewith.

[0066] Nucleic acid molecules encoding any of the LCDR2 amino acid sequences listed in Table 4 are provided herein, and in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCDR2 nucleic acid sequences listed in Table 5, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity therewith.

[0067] Nucleic acid molecules encoding any of the LCDR3 amino acid sequences listed in Table 4 are provided herein, and in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCDR3 nucleic acid sequences listed in Table 5, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity therewith.

[0068] A nucleic acid molecule encoding HCVR is provided herein, which comprises a set of three CDRs (i.e., HCDR1-HCDR2-HCDR3), the set of HCDR1-HCDR2-HCDR3 amino acid sequences as defined by one of the exemplary anti-CD28 antibodies listed in Table 4.

[0069] A nucleic acid molecule encoding LCVR is provided herein, which comprises a set of three CDRs (i.e., LCDR1-LCDR2-LCDR3), the set of amino acid sequences of LCDR1-LCDR2-LCDR3 as defined by one of the exemplary anti-CD28 antibodies listed in Table 4.

[0070] Nucleic acid molecules encoding both HCVR and LCVR are provided herein, where HCVR comprises an amino acid sequence of any of the HCVR amino acid sequences listed in Table 4, and LCVR comprises an amino acid sequence of any of the LCVR amino acid sequences listed in Table 4. In certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCVR nucleic acid sequences listed in Table 5, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto, and a polynucleotide sequence selected from any of the LCVR nucleic acid sequences listed in Table 5, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto. In certain embodiments, the nucleic acid molecule encodes both HCVR and LCVR, and both HCVR and LCVR are derived from the same anti-CD28 antibody listed in Table 4.

[0071] This specification provides recombinant expression vectors capable of expressing polypeptides comprising a heavy chain variable region or a light chain variable region of an anti-CD28 antibody. For example, this disclosure includes recombinant expression vectors comprising any of the nucleic acid molecules described above, i.e., nucleic acid molecules encoding any of the HCVR sequence, LCVR sequence, and / or CDR sequence as listed in Table 4. Host cells into which such vectors have been introduced, as well as methods for producing an antibody or a portion thereof by culturing host cells under conditions that enable the production of an antibody or antibody fragment, and methods for recovering the antibody and antibody fragment thus produced, are also included within the scope of this disclosure.

[0072] Anti-CD28 antibodies having modified glycosylation patterns are provided herein. In some embodiments, for example, modifications to remove undesirable glycosylation sites, i.e., antibodies lacking the fucose portion present on the oligosaccharide chain, may be useful to enhance antibody-dependent cell-mediated cytotoxicity (ADCC) function (see Shield et al. (2002) JBC277:26733). In other applications, galactosylation modifications can be made to modify complement-dependent cell-mediated cytotoxicity (CDC) activity.

[0073] This specification provides pharmaceutical compositions comprising a recombinant human antibody or fragment thereof that specifically binds to CD28 and a pharmaceutically acceptable carrier. In relevant embodiments, this disclosure features compositions that are combinations of an anti-CD28 antibody and a second therapeutic agent. In one embodiment, the second therapeutic agent is any agent that is advantageously combined with the anti-CD28 antibody. Exemplary agents that may be advantageously combined with the anti-CD28 antibody include, but are not limited to, other agents that activate or stimulate the activation of immune cells (including other antibodies or their antigen-binding fragments) and / or agents that do not directly bind to CD28 but promote an antitumor response. Additional combination therapies and combinations involving the anti-CD28 antibody of this disclosure are disclosed elsewhere in this specification.

[0074] In yet another embodiment, a therapeutic method for stimulating T cell activation using an anti-CD28 antibody or an antigen-binding moiety of an antibody is provided herein, the therapeutic method comprising administering a therapeutically effective amount of a pharmaceutical composition comprising an antibody or antigen-binding moiety of an anti-CD28 antibody to a subject requiring such treatment. The disorder to be treated is any disease or condition that is improved, remitted, inhibited, or prevented by stimulation of CD28 activity or signaling.

[0075] Bispecific antibodies containing anti-CD38 and anti-CD28 antigen-binding domains In another embodiment, the disclosure provides a bispecific antigen-binding molecule that binds to CD28 and a target antigen. In yet another embodiment, the disclosure provides a bispecific antigen-binding molecule that binds to CD38 and a co-stimulatory molecule. According to certain exemplary embodiments, the bispecific antigen-binding molecule binds to CD38 and CD28, and such a bispecific antigen-binding molecule is also referred to herein as an “anti-CD38 / anti-CD28 bispecific molecule”.

[0076] The anti-CD38 portion of the anti-CD38 / anti-CD28 bispecific molecule is useful for targeting tumor cells expressing CD38 (e.g., plasma cells), while the anti-CD28 portion of the bispecific molecule is useful for providing co-stimulation of T cells activated by homologous MHC peptides or tumor-targeting CD3 bispecific antibodies. The simultaneous binding of CD38 on tumor cells and CD28 on T cells promotes direct killing (cytolysis) of target tumor cells by activated T cells. Therefore, the anti-CD38 / anti-CD28 bispecific molecule of the present invention is particularly useful in the treatment of diseases and disorders associated with or caused by CD38-expressing tumors (e.g., lymphoma, leukemia, and multiple myeloma).

[0077] The bispecific antigen-binding molecules provided herein include a first antigen-binding domain that specifically binds to human CD38 and a second antigen-binding domain that specifically binds to CD28. This disclosure includes anti-CD38 / anti-CD28 bispecific molecules (e.g., bispecific antibodies), each antigen-binding domain including a heavy chain variable region (HCVR) paired with a light chain variable region (LCVR). In certain exemplary embodiments of this disclosure, the anti-CD38 antigen-binding domain and the anti-CD28 antigen-binding domain each include different distinct HCVRs paired with a common LCVR. For example, as illustrated in Example 4 of this specification, a bispecific antibody is constructed comprising a first antigen-binding domain that specifically binds to CD38 (the first antigen-binding domain contains an HCVR / LCVR pair derived from an anti-CD38 antibody) and a second antigen-binding domain that specifically binds to CD28 (the second antigen-binding domain contains an HCVR derived from anti-CD28 paired with an LCVR derived from an anti-CD38 antibody (e.g., the same LCVR contained in the anti-CD38 antigen-binding domain)). In such embodiments, the first and second antigen-binding domains contain separate anti-CD38 and anti-CD28 HCVRs but share a common anti-CD38 LCVR.

[0078] This specification provides an anti-CD38 / anti-CD28 bispecific molecule in which a first antigen-binding domain that specifically binds to CD38 comprises one of the HCVR amino acid sequences listed in Table 1. The first antigen-binding domain that specifically binds to CD38 may also comprise one of the LCVR amino acid sequences listed in Table 1. According to certain embodiments, the first antigen-binding domain that specifically binds to CD38 comprises one of the HCVR / LCVR amino acid sequence pairs listed in Table 1. This disclosure also provides an anti-CD38 / anti-CD28 bispecific molecule in which a first antigen-binding domain that specifically binds to CD38 comprises one of the heavy chain CDR1-CDR2-CDR3 amino acid sequences listed in Table 1 and / or one of the light chain CDR1-CDR2-CDR3 amino acid sequences listed in Table 1.

[0079] According to a particular embodiment, the Disclosure provides an anti-CD38 / anti-CD28 bispecific molecule in which a first antigen-binding domain that specifically binds to CD38 comprises a heavy chain variable region (HCVR) having an amino acid sequence selected from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 32, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0080] This specification provides an anti-CD38 / anti-CD28 bispecific molecule in which a first antigen-binding domain that specifically binds to CD38 comprises a light chain variable region (LCVR) having an amino acid sequence selected from the group consisting of SEQ ID NO: 18 and SEQ ID NO: 48, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0081] This specification provides an anti-CD38 / anti-CD28 bispecific molecule in which the first antigen-binding domain, which specifically binds to CD38, comprises an HCVR and LCVR (HCVR / LCVR) amino acid sequence pair selected from the group consisting of SEQ ID NOs: 2 / 18 and 32 / 48.

[0082] This specification provides an anti-CD38 / anti-CD28 bispecific molecule in which a first antigen-binding domain that specifically binds to CD38 comprises a heavy chain CDR3 (HCDR3) domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 8 and 38, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity, and a light chain CDR3 (LCDR3) domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 24 and 54, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0083] In certain embodiments, the first antigen-binding domain that specifically binds to CD38 comprises an HCDR3 / LCDR3 amino acid sequence pair selected from the group consisting of SEQ ID NOs: 8 / 24 and 38 / 54.

[0084] This specification provides an anti-CD38 / anti-CD28 bispecific molecule wherein the first antigen-binding domain that specifically binds to CD38 comprises a heavy chain CDR1 (HCDR1) domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 and 34, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity, and an amino acid sequence selected from the group consisting of SEQ ID NOs: 6 and 36, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. The material comprises a heavy chain CDR2 (HCDR2) domain having a sequence, a light chain CDR1 (LCDR1) domain having an amino acid sequence selected from the group consisting of SEQ ID NOs. 20 and 50, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity, and a light chain CDR2 (LCDR2) domain having an amino acid sequence selected from the group consisting of SEQ ID NOs. 22 and 52, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0085] A particular, non-limiting, exemplary anti-CD38 / anti-CD28 bispecific antigen-binding molecule of the present invention comprises an HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domain, each having an amino acid sequence selected from the group consisting of SEQ ID NOs: 4-6-8-20-22-24 (e.g., bsAb6031) and 34-36-38-50-52-54 (e.g., bsAb7945), and includes a first antigen-binding domain that specifically binds to CD38.

[0086] This specification provides an anti-CD38 / anti-CD28 bispecific molecule in which a second antigen-binding domain that specifically binds to CD28 comprises one of the HCVR amino acid sequences listed in Table 4. The second antigen-binding domain that specifically binds to CD28 may also comprise one of the LCVR amino acid sequences listed in Table 4. According to certain embodiments, the second antigen-binding domain that specifically binds to CD28 comprises one of the HCVR / LCVR amino acid sequence pairs listed in Table 4. This disclosure also provides an anti-CD38 / anti-CD28 bispecific molecule in which a second antigen-binding domain that specifically binds to CD28 comprises one of the heavy chain CDR1-CDR2-CDR3 amino acid sequences listed in Table 4 and / or one of the light chain CDR1-CDR2-CDR3 amino acid sequences listed in Table 4.

[0087] According to certain embodiments, the Specified provides an anti-CD38 / anti-CD28 bispecific molecule in which a second antigen-binding domain that specifically binds to CD28 comprises a heavy chain variable region (HCVR) having an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NO: 10 and SEQ ID NO: 40, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0088] This specification provides an anti-CD38 / anti-CD28 bispecific molecule in which a second antigen-binding domain that specifically binds to CD28 comprises a light chain variable region (LCVR) having an amino acid sequence selected from the group consisting of SEQ ID NO: 18 and SEQ ID NO: 48, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0089] This specification provides an anti-CD38 / anti-CD28 bispecific molecule in which a second antigen-binding domain that specifically binds to CD28 comprises an HCVR and LCVR (HCVR / LCVR) amino acid sequence pair selected from the group consisting of SEQ ID NOs: 10 / 18 and 40 / 48.

[0090] This specification provides an anti-CD38 / anti-CD28 bispecific molecule in which a second antigen-binding domain that specifically binds to CD28 comprises a heavy chain CDR3 (HCDR3) domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 16 and 46, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity, and a light chain CDR3 (LCDR3) domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 24 and 54, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0091] In certain embodiments, the second antigen-binding domain that specifically binds to CD28 includes an HCDR3 / LCDR3 amino acid sequence pair selected from the group consisting of SEQ ID NOs: 16 / 24 and 46 / 54.

[0092] This specification provides an anti-CD38 / anti-CD28 bispecific molecule, wherein the second antigen-binding domain that specifically binds to CD28 comprises a heavy chain CDR1 (HCDR1) domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 12 and 42, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity, and an amino acid sequence selected from the group consisting of SEQ ID NOs: 14 and 44, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. It comprises a heavy chain CDR2 (HCDR2) domain having a sequence, a light chain CDR1 (LCDR1) domain having an amino acid sequence selected from the group consisting of SEQ ID NOs. 20 and 50, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity, and a light chain CDR2 (LCDR2) domain having an amino acid sequence selected from the group consisting of SEQ ID NOs. 22 and 52, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0093] Certain non-limiting exemplary anti-CD38 / anti-CD28 bispecific antigen-binding molecules provided herein include an HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domain, each comprising a second antigen-binding domain that specifically binds to CD28 and has an amino acid sequence selected from the group consisting of SEQ ID NOs: 12-14-16-20-22-24 (e.g., bsAb6031) and 42-44-46-50-52-54 (e.g., bsAb7945).

[0094] In the embodiments provided herein, multispecific (e.g., bispecific) antigen-binding molecules (e.g., antibodies or their antigen-binding fragments) that bind to CD38 and CD28 include the following: (1) Anti-CD38 coupling arm including the following: HCDR1 containing amino acid sequence: GFTFDDYA (SEQ ID NO: 4, or its variant), HCDR2 containing amino acid sequence: ISWKSDNI (SEQ ID NO: 6, or its variant), HCDR3 containing amino acid sequence: AKALGGWKFDYYYGMDV (SEQ ID NO: 8, or its variant), LCDR1 containing amino acid sequence: QSISSY (SEQ ID NO: 20, or its variant), LCDR2 containing amino acid sequence: AAS (SEQ ID NO: 22, or its variant), and LCDR3 containing amino acid sequence: QQSYSTPPIT (SEQ ID NO: 24, or its variant), and Anti-CD28 coupling arm including the following: HCDR1 containing amino acid sequence: GFTFSRNN (SEQ ID NO: 12, or its variant), HCDR2 containing amino acid sequence: ISSNGGRT (SEQ ID NO: 14, or its variant), HCDR3 containing amino acid sequence: TRDDELLSFDY (SEQ ID NO: 16, or its variant), LCDR1 containing amino acid sequence: QSISSY (SEQ ID NO: 20, or its variant), LCDR2 containing amino acid sequence: AAS (SEQ ID NO: 22, or its variant), and LCDR3 containing amino acid sequence: QQSYSTPPIT (SEQ ID NO: 24, or its variant); (2) Anti-CD38 coupling arm including the following: HCDR1 containing amino acid sequence:GGPFRSSS (SEQ ID NO: 34, or its variant), HCDR2 containing amino acid sequence:IIPILGKT (SEQ ID NO: 36, or its variant), HCDR3 containing amino acid sequence:VRGSSLFDY (SEQ ID NO: 38, or its variant), LCDR1 containing amino acid sequence:QSVSSSY (SEQ ID NO: 50, or its variant), LCDR2 containing amino acid sequence:GAS (SEQ ID NO: 52, or its variant), and LCDR3 containing amino acid sequence:QQYGSSPWT (SEQ ID NO: 54, or its variant), and Anti-CD28 coupling arm including the following: HCDR1 containing amino acid sequence: GGSISSYY (SEQ ID NO: 42 or its variant), HCDR2 containing amino acid sequence: IYYSGIT (SEQ ID NO: 44 or its variant), HCDR3 containing amino acid sequence: ARWGVRRDYYYYGMDV (SEQ ID NO: 46 or its variant), LCDR1 containing amino acid sequence: QSVSSSY (SEQ ID NO: 50 or its variant), LCDR2 containing amino acid sequence: GAS (SEQ ID NO: 52 or its variant), and LCDR3 containing amino acid sequence: QQYGSSPWT (SEQ ID NO: 54 or its variant); (3) Anti-CD38 coupling arm including the following: HCVR1 containing the amino acid sequence:EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGISWKSDNIGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCAKALGGWKFDYYYGMDVWGQGTTVTVSS (SEQ ID NO: 2 or its variant), and LCVR1 containing the amino acid sequence:DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPITFGQGTRLEIK (SEQ ID NO: 18 or its variant), and Anti-CD28 coupling arm including the following: HCVR1 containing the amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGFTFSRNNMHWVRQAPGKGLEYVSGISSNGGRTYYADSVKGRFTISRDNSKNTLYLQMGGLRAADMAVYFCTRDDELLSFDYWGQGTLVTVSS (SEQ ID NO: 10, or its variant), and LCVR1 containing the amino acid sequence: DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPITFGQGTRLEIK (SEQ ID NO: 18, or its variant); (4) Anti-CD38 coupling arm including the following: HCVR1 containing amino acid sequence: QVQLVQSGAEVKKPGSSVKVSCKASGGPFRSSSFSWVRQAPGQGLEWMGGIIPILGKTNYAQKFQGRITIVTDESTTTVYMELSSLRSEDTAVFYCVRGSSLFDYWGQGTLVTVSS (SEQ ID NO: 32, or its variant), and LCVR1 containing EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIK (SEQ ID NO: 48, or its variant), Anti-CD28 coupling arm including the following: HCVR1 containing amino acid sequence: QVQLQESGPGLVKPSETLSLTCTVSGGSISSYYWSWIRQPPGKGLEWIGYIYYSGITHYNPSLKSRVTISVDTSKIQFSLKLSSVTAADTAVYYCARWGVRRDYYYYGMDVWGQGTTVTVSS (SEQ ID NO: 40, or its variant), and LCVR1 containing EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIK (SEQ ID NO: 48, or its variant); (5) Anti-CD38 coupling arm including the following: Amino acid sequence EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGISWKSDNIGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCAKALGGWKFDYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPA A heavy chain containing PPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK* (SEQ ID NO: 26, or its variant), and A light chain containing the amino acid sequence DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPITFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC* (SEQ ID NO: 30, or its variant), and Anti-CD28 coupling arm including the following: Amino acid sequence EVQLVESGGGLVQPGGSLRLSCAASGFTFSRNNMHWVRQAPGKGLEYVSGISSNGGRTYYADSVKGRFTISRDNSKNTLYLQMGGLRAADMAVYFCTRDDELLSFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPPV A heavy chain containing AGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNRFTQKSLSLSPGK* (SEQ ID NO: 28, or its variant), and A light chain containing the amino acid sequence DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPITFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC* (SEQ ID NO: 30, or its variant); or (6) Anti-CD38 coupling arm including the following: Amino acid sequence QVQLVQSGAEVKKPGSSVKVSCKASGGPFRSSSFSWVRQAPGQGLEWMGGIIPILGKTNYAQKFQGRITIVTDESTTTVYMELSSLRSEDTAVFYCVRGSSLFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPPVA A heavy chain containing GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK* (SEQ ID NO: 56, or its variant), and A light chain containing the amino acid sequence EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC* (SEQ ID NO: 60, or its variant), and Anti-CD28 coupling arm including the following: Amino acid sequence QVQLQESGPGLVKPSETLSLTCTVSGGSISSYYWSWIRQPPGKGLEWIGYIYYSGITHYNPSLKSRVTISVDTSKIQFSLKLSSVTAADTAVYYCARWGVRRDYYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAP A heavy chain containing PVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNRFTQKSLSLSPGK* (SEQ ID NO: 58, or its variant), and A light chain containing the amino acid sequence EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC* (SEQ ID NO: 60, or its variant).

[0095] In the embodiments provided herein, multispecific (e.g., bispecific) antigen-binding molecules (e.g., antibodies or their antigen-binding fragments) that bind to CD38 and CD28 include the following: (1) Anti-CD38 coupling arm including the following: HCDR1 containing amino acid sequence: GFTFDDYA (SEQ ID NO: 4, or its variant), HCDR2 containing amino acid sequence: ISWKSDNI (SEQ ID NO: 6, or its variant), HCDR3 containing amino acid sequence: AKALGGWKFDYYYGMDV (SEQ ID NO: 8, or its variant), LCDR1 containing amino acid sequence: QSISSY (SEQ ID NO: 20, or its variant), LCDR2 containing amino acid sequence: AAS (SEQ ID NO: 22, or its variant), and LCDR3 containing amino acid sequence: QQSYSTPPIT (SEQ ID NO: 24, or its variant), and Anti-CD28 coupling arm including the following: HCDR1 containing amino acid sequence: GGSISSYY (SEQ ID NO: 42 or its variant), HCDR2 containing amino acid sequence: IYYSGIT (SEQ ID NO: 44 or its variant), HCDR3 containing amino acid sequence: ARWGVRRDYYYYGMDV (SEQ ID NO: 46 or its variant), LCDR1 containing amino acid sequence: QSVSSSY (SEQ ID NO: 50 or its variant), LCDR2 containing amino acid sequence: GAS (SEQ ID NO: 52 or its variant), and LCDR3 containing amino acid sequence: QQYGSSPWT (SEQ ID NO: 54 or its variant); (2) Anti-CD38 coupling arm including the following: HCDR1 containing amino acid sequence:GGPFRSSS (SEQ ID NO: 34, or its variant), HCDR2 containing amino acid sequence:IIPILGKT (SEQ ID NO: 36, or its variant), HCDR3 containing amino acid sequence:VRGSSLFDY (SEQ ID NO: 38, or its variant), LCDR1 containing amino acid sequence:QSVSSSY (SEQ ID NO: 50, or its variant), LCDR2 containing amino acid sequence:GAS (SEQ ID NO: 52, or its variant), and LCDR3 containing amino acid sequence:QQYGSSPWT (SEQ ID NO: 54, or its variant), and Anti-CD28 coupling arm including the following: HCDR1 containing amino acid sequence: GFTFSRNN (SEQ ID NO: 12, or its variant), HCDR2 containing amino acid sequence: ISSNGGRT (SEQ ID NO: 14, or its variant), HCDR3 containing amino acid sequence: TRDDELLSFDY (SEQ ID NO: 16, or its variant), LCDR1 containing amino acid sequence: QSISSY (SEQ ID NO: 20, or its variant), LCDR2 containing amino acid sequence: AAS (SEQ ID NO: 22, or its variant), and LCDR3 containing amino acid sequence: QQSYSTPPIT (SEQ ID NO: 24, or its variant); (3) Anti-CD38 coupling arm including the following: HCVR1 containing the amino acid sequence:EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGISWKSDNIGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCAKALGGWKFDYYYGMDVWGQGTTVTVSS (SEQ ID NO: 2 or its variant), and LCVR1 containing the amino acid sequence:DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPITFGQGTRLEIK (SEQ ID NO: 18 or its variant), and Anti-CD28 coupling arm including the following: HCVR1 containing amino acid sequence: QVQLQESGPGLVKPSETLSLTCTVSGGSISSYYWSWIRQPPGKGLEWIGYIYYSGITHYNPSLKSRVTISVDTSKIQFSLKLSSVTAADTAVYYCARWGVRRDYYYYGMDVWGQGTTVTVSS (SEQ ID NO: 40, or its variant), and LCVR1 containing EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIK (SEQ ID NO: 48, or its variant); (4) Anti-CD38 coupling arm including the following: HCVR1 containing amino acid sequence: QVQLVQSGAEVKKPGSSVKVSCKASGGPFRSSSFSWVRQAPGQGLEWMGGIIPILGKTNYAQKFQGRITIVTDESTTTVYMELSSLRSEDTAVFYCVRGSSLFDYWGQGTLVTVSS (SEQ ID NO: 32, or its variant), and LCVR1 containing EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIK (SEQ ID NO: 48, or its variant), Anti-CD28 coupling arm including the following: HCVR1 containing the amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGFTFSRNNMHWVRQAPGKGLEYVSGISSNGGRTYYADSVKGRFTISRDNSKNTLYLQMGGLRAADMAVYFCTRDDELLSFDYWGQGTLVTVSS (SEQ ID NO: 10, or its variant), and LCVR1 containing the amino acid sequence: DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPITFGQGTRLEIK (SEQ ID NO: 18, or its variant); (5) Anti-CD38 coupling arm including the following: Amino acid sequence EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGISWKSDNIGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCAKALGGWKFDYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPA A heavy chain containing PPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK* (SEQ ID NO: 26, or its variant), and A light chain containing the amino acid sequence DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPITFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC* (SEQ ID NO: 30, or its variant), and Anti-CD28 coupling arm including the following: Amino acid sequence QVQLQESGPGLVKPSETLSLTCTVSGGSISSYYWSWIRQPPGKGLEWIGYIYYSGITHYNPSLKSRVTISVDTSKIQFSLKLSSVTAADTAVYYCARWGVRRDYYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAP A heavy chain containing PVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNRFTQKSLSLSPGK* (SEQ ID NO: 58, or its variant), and A light chain containing the amino acid sequence EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC* (SEQ ID NO: 60, or its variant); or (6) Anti-CD38 coupling arm including the following: Amino acid sequence QVQLVQSGAEVKKPGSSVKVSCKASGGPFRSSSFSWVRQAPGQGLEWMGGIIPILGKTNYAQKFQGRITIVTDESTTTVYMELSSLRSEDTAVFYCVRGSSLFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPPVA A heavy chain containing GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK* (SEQ ID NO: 56, or its variant), and A light chain containing the amino acid sequence EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC* (SEQ ID NO: 60, or its variant), and Anti-CD28 coupling arm including the following: Amino acid sequence EVQLVESGGGLVQPGGSLRLSCAASGFTFSRNNMHWVRQAPGKGLEYVSGISSNGGRTYYADSVKGRFTISRDNSKNTLYLQMGGLRAADMAVYFCTRDDELLSFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPPV A heavy chain containing AGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNRFTQKSLSLSPGK* (SEQ ID NO: 28, or its variant), and A light chain containing the amino acid sequence DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPITFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC* (SEQ ID NO: 30, or its variant).

[0096] In another embodiment, nucleic acid molecules are provided herein that encode any of the HCVR, LCVR, or CDR sequences of the anti-CD38 / anti-CD28 bispecific antigen-binding molecules disclosed herein, comprising nucleic acid molecules containing the polynucleotide sequences listed in Table 2 herein, and nucleic acid molecules containing the polynucleotide sequences listed in Table 5 herein, in any functional combination or arrangement thereof. Recombinant expression vectors carrying the nucleic acids of the present invention and host cells into which such vectors have been introduced are also encompassed by the present invention, as are methods for producing antibodies by culturing host cells under conditions that enable antibody production and recovering the produced antibodies.

[0097] The present invention includes an anti-CD38 / anti-CD28 bispecific antigen-binding molecule, wherein any of the aforementioned antigen-binding domains that specifically bind to CD38 are combined with, linked to, or associated with any of the aforementioned antigen-binding domains that specifically bind to CD28 to form a bispecific antigen-binding molecule that binds CD38 and CD28.

[0098] Anti-CD38 / anti-CD28 bispecific antigen-binding molecules having modified glycosylation patterns are provided herein. In some applications, for example, to enhance antibody-dependent cell-mediated cytotoxicity (ADCC) function, modifications to remove undesirable glycosylation sites, i.e., antibodies lacking the fucose portion present on the oligosaccharide chain, may be useful (see Shield et al. (2002) JBC277:26733). In other applications, galactosylation modifications can be made to modify complement-dependent cell-mediated cytotoxicity (CDC) activity.

[0099] In another embodiment, this specification provides a pharmaceutical composition comprising an anti-CD38 / anti-CD28 bispecific antigen-binding molecule disclosed herein and a pharmaceutically acceptable carrier. In a related embodiment, the present invention features a composition which is a combination of an anti-CD38 / anti-CD28 bispecific antigen-binding molecule and a second therapeutic agent. In one embodiment, the second therapeutic agent is any agent which can be advantageously combined with the anti-CD38 / anti-CD28 bispecific antigen-binding molecule. Exemplary agents which can be advantageously combined with the anti-CD38 / anti-CD28 bispecific antigen-binding molecule are described in detail elsewhere in this specification.

[0100] In yet another embodiment, a therapeutic method for targeting / killing tumor cells expressing CD38 using the anti-CD38 / anti-CD28 bispecific antigen-binding molecule of the present invention is provided herein, the therapeutic method comprising administering a pharmaceutical composition containing a therapeutically effective amount of the anti-CD38 / anti-CD28 bispecific antigen-binding molecule to a subject requiring it.

[0101] This disclosure also includes the use of anti-CD38 / anti-CD28 bispecific antigen-binding molecules provided herein in the manufacture of pharmaceuticals for the treatment of diseases or disorders related to or caused by CD38 expression.

[0102] Other embodiments will become apparent from the examination of the detailed description below. [Brief explanation of the drawing]

[0102] [Figure 1] This shows cytotoxicity, T cell activation, T cell proliferation, and cytokine release in H929 tumor cells after treatment with the costimulatory anti-CD38×CD28 bispecific antibodies bsAb6031 and bsAb7954. [Figure 2] This shows cytotoxicity, T cell activation, T cell proliferation, and cytokine release in H929 tumor cells after treatment with the costimulatory anti-CD38×CD28 bispecific antibodies bsAb6031 and bsAb7954. [Figure 3]This shows cytotoxicity, T cell activation, T cell proliferation, and cytokine release in H929 tumor cells after treatment with the costimulatory anti-CD38×CD28 bispecific antibodies bsAb6031 and bsAb7954. [Figure 4-1] This shows cytotoxicity, T cell activation, T cell proliferation, and cytokine release in H929 tumor cells after treatment with the costimulatory anti-CD38×CD28 bispecific antibodies bsAb6031 and bsAb7954. [Figure 4-2] Continuation of Figure 4-1. [Figure 5] The in vivo antitumor activity of the costimulatory anti-CD38×CD28 bispecific antibodies bsAb6031 and bsAb7954, either alone or in combination with BCMA×CD3bsAb, is demonstrated. [Figure 6-1] The in vivo antitumor activity of the costimulatory anti-CD38×CD28 bispecific antibodies bsAb6031 and bsAb7954, either alone or in combination with BCMA×CD3bsAb, is demonstrated. [Figure 6-2] Continuation of Figure 6-1. [Figure 7] The in vivo antitumor activity of the costimulatory anti-CD38×CD28 bispecific antibodies bsAb6031 and bsAb7954, either alone or in combination with BCMA×CD3bsAb, is demonstrated. [Figure 8] The in vivo antitumor activity of the costimulatory anti-CD38×CD28 bispecific antibodies bsAb6031 and bsAb7954, either alone or in combination with BCMA×CD3bsAb, is demonstrated. [Figure 9] The in vivo antitumor activity of the costimulatory anti-CD38×CD28 bispecific antibodies bsAb6031 and bsAb7954, either alone or in combination with BCMA×CD3bsAb, is demonstrated. [Figure 10] The in vivo antitumor activity of the costimulatory anti-CD38×CD28 bispecific antibodies bsAb6031 and bsAb7954, either alone or in combination with BCMA×CD3bsAb, is demonstrated. [Modes for carrying out the invention]

[0103] Before describing the present invention, it should be understood that the present invention is not limited to the specific methods and experimental conditions described, and therefore the methods and conditions may vary. Since the scope of the present invention is limited only by the appended claims, it should also be understood that the terms used herein are used solely to describe specific embodiments and are not intended to limit them.

[0104] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which the present invention pertains. Where used herein, the term “about” means that, when used in reference to a specific numerical value, the value may vary by up to 1% or less from the value mentioned. For example, where used herein, the expression “about 100” includes 99 and 101, as well as all values ​​in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0105] Any methods and materials similar or equivalent to those described herein may be used in the practice or testing of the present invention, but preferred methods and materials are described herein. All patents, patent applications, and non-patent publications referenced herein are incorporated herein by reference in their entirety.

[0106] Definition of Terms As used herein, "expressed CD28" refers to an antigen expressed as a homodimer on T cells. Human CD28 comprises the amino acid sequence described in SEQ ID NO: 61 (human CD28 extracellular domain (N19-P152).mFc) and / or has the amino acid sequence described in NCBI Commission No. NM_006139.3 and / or has the amino acid sequence of human CD28>NP_006130.1 T cell-specific surface glycoprotein CD28 isoform 1 precursor (SEQ ID NO: 63).

[0107] Human CD28 extracellular domain (N19-P152).mFc (immunogen) amino acid sequence [ka] All references to proteins, polypeptides, and protein fragments herein are intended to refer to the human versions of the respective proteins, polypeptides, or protein fragments unless explicitly specified as being of non-human origin. Therefore, the expression "CD28" means human CD28 unless otherwise specified, for example, "mouse CD28" or "monkey CD28," indicating a non-human origin.

[0108] As used herein, “CD28-binding antibody” or “anti-CD28 antibody” includes an antibody and its antigen-binding fragment that specifically recognize a single CD28 unit, and an antibody and its antigen-binding fragment that specifically recognize a dimeric complex of two CD28 subunits. The antibodies and antigen-binding fragments of the present invention can bind to soluble CD28 and / or cell surface-expressed CD28. Soluble CD28 includes the native CD28 protein, as well as recombinant CD28 protein variants that lack a transmembrane domain or are otherwise not associated with the cell membrane, such as monomeric and dimeric CD28 constructs.

[0109] As used herein, the expression “cell surface expressed CD28” means one or more CD28 proteins expressed in vitro or in vivo on the surface of a cell such that at least a portion of the CD28 protein is exposed to the extracellular side of the cell membrane and is accessible to the antigen-binding portion of an antibody. “Cell surface expressed CD28” includes CD28 proteins associated with functional T cell receptors on the cell membrane. The expression “cell surface expressed CD28” includes CD28 proteins expressed on the surface of a cell as part of a homodimer. “Cell surface expressed CD28” includes, or may consist of, CD28 proteins expressed on the surface of a cell that normally expresses the CD28 protein. Alternatively, “cell surface expressed CD28” includes, or may consist of, CD28 proteins expressed on the surface of a cell that does not normally express human CD28 on its surface but has been artificially engineered to express CD28 on its surface.

[0110] As used herein, the expression "CD38" also refers to a glycoprotein known as cyclic ADP ribose hydrolase and expressed on malignant cells. CD38 plays a central role in the regulation of intracellular calcium levels. The protein has an N-terminal cytoplasmic tail, a single transmembrane domain, and a C-terminal extracellular region with four N-glycosylation sites.

[0111] As used herein, "antibody that binds to CD38" or "anti-CD38 antibody" includes an antibody that specifically recognizes CD38 and its antigen-binding fragments.

[0112] The term "antigen-binding molecule" includes antibodies and antigen-binding fragments of antibodies, and includes, for example, bispecific antibodies.

[0113] As used herein, the term "antibody" means any antigen-binding molecule or molecular complex that includes at least one complementarity-determining region (CDR) that specifically binds to or interacts with a particular antigen (e.g., CD38 or CD28). The term "antibody" includes immunoglobulin molecules comprising four polypeptide chains interconnected by disulfide bonds, i.e., two heavy (H) chains and two light (L) chains, and multimers thereof (e.g., IgM). The term "antibody" also includes immunoglobulin molecules consisting of four polypeptide chains interconnected by disulfide bonds, i.e., two heavy (H) chains and two light (L) chains. Each heavy chain includes a heavy chain variable region (abbreviated herein as HCVR or V H and omitted) and a heavy chain constant region. The heavy chain constant region includes three domains, C H 1, C H 2, and C H 3. Each light chain includes a light chain variable region (abbreviated herein as LCVR or V L and omitted) and a light chain constant region. The light chain constant region includes one domain (C L 1). The V H region and the V LThe region can be further subdivided into a highly variable region called the Complementarity Determination Region (CDR), which is interspersed with a relatively conserved region called the Framework Region (FR). H and V L It consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In different embodiments disclosed herein, the FRs of the anti-CD38 antibody or anti-CD28 antibody (or its antigen-binding portion) may be identical to the human germline sequence or may be naturally or artificially modified. The amino acid consensus sequence may be defined based on a parallel analysis of two or more CDRs.

[0114] As used herein, the term “antibody” also includes the antigen-binding fragment of a complete antibody molecule. As used herein, the terms “antigen-binding portion” of an antibody, “antigen-binding fragment” of an antibody, etc., include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. Antigen-binding fragments of antibodies can be obtained from a complete antibody molecule using any suitable standard method, such as proteolytic digestion or recombinant genetic engineering techniques, which involve the manipulation and expression of DNA encoding an antibody variable region and optionally a constant domain. Such DNA is known and / or readily available, for example, from commercially available sources, DNA libraries (including, for example, phage antibody libraries), or can be synthesized. DNA can be manipulated by using chemical or molecular biological techniques to sequence it and, for example, to position one or more variable domains and / or constant domains in a suitable configuration, or to introduce codons, create cysteine ​​residues, or modify, add, or delete amino acids.

[0115] Non-limiting examples of antigen-binding fragments include (i) Fab fragments, (ii) F(ab')2 fragments, (iii) Fd fragments, (iv) Fv fragments, (v) single-chain Fv(scFv) molecules, (vi) dAb fragments, and (vii) minimal recognition units consisting of amino acid residues mimicking the hypervariable region of an antibody (e.g., isolated complementarity-determining regions (CDRs) such as the CDR3 peptide), or constrained FR3-CDR3-FR4 peptides. Other manipulated molecules such as domain-specific antibodies, single-domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-implanted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunotherapies (SMIPs), and shark variable IgNAR domains are also included in the expression “antigen-binding fragment” as used herein.

[0116] The antigen-binding fragment of an antibody typically contains at least one variable domain. The variable domain may be of any size or amino acid composition, and generally contains at least one CDR adjacent to or in-frame one or more framework sequences. L V associating with the domain H In an antigen-binding fragment having a domain, V H Domain and V L Domains can be arranged relative to each other in any suitable configuration. For example, the variable region is a dimer, V H -V H , V H -V L or V L -V L It may contain a dimer. Alternatively, the antigen-binding fragment of the antibody may be a monomer V H or V L It may contain a domain.

[0117] In certain embodiments, the antigen-binding fragment of the antibody may contain at least one variable domain covalently bound to at least one constant domain. Non-limiting exemplary configurations of variable and constant domains that may be found in the antigen-binding fragment of the antibody of the present invention include (i)VH -C H 1. (ii)V H -C H 2, (iii)V H -C H 3, (iv)V H -C H 1-C H 2. (v)V H -C H 1-C H 2-C H 3. (vi)V H -C H 2-C H 3. (vii)V H -C L (viii)V L -C H 1. (ix)V L -C H 2, (x)V L -C H 3. (xi)V L -C H 1-C H 2. (xii)V L -C H 1-C H 2-C H 3. (xiii)V L -C H 2-C H 3, and (xiv)V L -C L Examples include: In any configuration of a variable domain and a constant domain, including any of the exemplary configurations listed above, the variable domain and the constant domain may be directly linked to each other, or they may be linked by a complete or partial hinge or linker region. The hinge region may consist of at least two (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids that result in a mobile or semi-mobile linkage between adjacent variable domains and / or constant domains in a single polypeptide molecule. Furthermore, the antigen-binding fragment of the antibody of the present invention may be linked to each other and / or one or more monomers V H Or V LNon-covalent association with the domain (e.g., via disulfide bonds) may include homodimers or heterodimers (or other polymers) of any of the variable domain configurations and constant domain configurations listed above.

[0118] Similar to fully antibody molecules, antigen-binding fragments may be monospecific or multispecific (e.g., bispecific). Multispecific antigen-binding fragments of antibodies typically comprise at least two distinct variable domains, each capable of specifically binding to a different antigen or to a different epitope on the same antigen. Any multispecific antibody format, including the exemplary bispecific antibody formats disclosed herein, can be adapted for use in the context of the antibody antigen-binding fragments of the present invention using routine techniques available in the art.

[0119] The antibodies of this disclosure may function via complement-dependent cytotoxicity (CDC) or antibody-dependent cell-mediated cytotoxicity (ADCC). "Complement-dependent cytotoxicity" (CDC) refers to the lysis of antigen-expressing cells by the antibodies of this disclosure in the presence of complement. "Antibody-dependent cell-mediated cytotoxicity" (ADCC) refers to a cell-mediated response in which nonspecific cytotoxic cells expressing the Fc receptor (FcR) (e.g., natural killer (NK) cells, neutrophils, and macrophages) recognize a bound antibody on target cells, thereby resulting in the lysis of the target cells. CDC and ADCC may be measured using assays known and available in the art (see, for example, U.S. Patents 5,500,362 and 5,821,337, and Clynes et al. (1998) Proc. Natl. Acad. Sci. (USA) 95:652-656). The constant region of an antibody is crucial in its ability to immobilize complement and mediate cell-dependent cytotoxicity. Therefore, antibody isotypes may be selected based on whether it is desirable for the antibody to mediate cytotoxicity.

[0120] In certain embodiments of the present invention, the anti-CD38 monospecific antibody, anti-CD28 monospecific antibody, or anti-CD38 × anti-CD28 bispecific antibody provided herein is a human antibody. The term "human antibody," as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the present invention may, for example, include amino acid residues in the CDR, particularly CDR3, that are not encoded by the human germline immunoglobulin sequence (e.g., mutations introduced by random or site-directed mutagenesis in vitro, or by somatic mutation in vivo). However, the term "human antibody," as used herein, is not intended to include antibodies in which a CDR sequence derived from the germline of another mammalian species, such as mouse, has been transplanted onto a human framework sequence.

[0121] In some embodiments, the antibodies of the present invention may be recombinant human antibodies. The term "recombinant human antibody," as used herein, is intended to include all human antibodies prepared, expressed, created, or isolated by genetic recombination means, such as antibodies expressed using a recombinant expression vector transduced into host cells (detailed below), recombinants, antibodies isolated from combinatorial human antibody libraries (detailed below), antibodies isolated from animals (e.g., mice) that are transgenic of human immunoglobulin genes (see, for example, Taylor et al. (1992) Nucl. Acids Res. 20:6287-6295), or antibodies prepared, expressed, created, or isolated by any other means, including splicing human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies are subjected to in vitro mutagenesis (or in vivo somatic mutagenesis if transgenic animals for human Ig sequences are used), so the V of the recombinant antibody H Region and V LThe amino acid sequence of the region is human germline V H Array and V L While derived from and related to the sequence, it is a sequence that cannot naturally exist in the in vivo human antibody germline repertoire.

[0122] Human antibodies can exist in two forms related to hinge heterogeneity. In one form, the immunoglobulin molecule contains four stable chain constructs of approximately 150–160 kDa, with the dimer held together by interchain heavy-chain disulfide bonds. In the second form, the dimer is not linked via interchain disulfide bonds, and the molecule of approximately 75–80 kDa consists of covalently bonded light and heavy chains (half-antibody). These forms have been extremely difficult to separate, even after affinity purification.

[0123] The frequency of occurrence of the second form in various intact IgG isotypes is due to structural differences related to the hinge region isotype of the antibody, but is not limited to these. A single amino acid substitution in the hinge region of the human IgG4 hinge can significantly reduce the occurrence of the second form to the level typically observed using the human IgG1 hinge (Angal et al. (1993) Molecular Immunology 30:105). This invention relates to the hinge, C H 2 regions or C H The antibodies include those having one or more mutations in three regions, and these mutations may be desirable, for example, in production, to improve the yield of a desired antibody type.

[0124] The antibodies of the present invention may be isolated antibodies. As used herein, “isolated antibody” means an antibody identified, isolated, and / or recovered from at least one component of its natural environment. For example, an antibody isolated or extracted from at least one component of a living organism, or from a tissue or cell in which such antibody naturally exists or is naturally produced, is an “isolated antibody” for the purposes of the present invention. Isolated antibodies also include antibodies in situ within recombinant cells. An isolated antibody is an antibody that has undergone at least one purification or isolation step. According to certain embodiments, an isolated antibody may not substantially contain other cellular material and / or chemical substances.

[0125] The present invention also includes one-arm antibodies that bind to CD38 or CD28. As used herein, “one-arm antibody” means an antigen-binding molecule comprising a single antibody heavy chain and a single antibody light chain. The one-arm antibodies of the present invention may comprise either the HCVR / LCVR or CDR amino acid sequences listed in Table 1 or Table 4.

[0126] The anti-CD38 antibodies, anti-CD28 antibodies, or anti-CD38 × anti-CD28 antibodies disclosed herein may contain one or more amino acid substitutions, insertions, and / or deletions in the framework and / or CDR region of the heavy and light chain variable domains compared to the corresponding germline sequence from which the antibody is derived. Such mutations can be readily identified by comparing the amino acid sequences disclosed herein with germline sequences available, for example, from publicly available antibody sequence databases. This disclosure includes antibodies derived from any of the amino acid sequences disclosed herein, and antigen-binding fragments thereof, in which one or more amino acids in one or more frameworks and / or CDR regions are mutated to corresponding residues in the germline sequence from which the antibody is derived, or to corresponding residues in another human germline sequence, or to conserved amino acid substitutions of corresponding germline residues (such sequence changes are collectively referred to herein as “germline mutations”). Those skilled in the art can readily produce many antibodies and antigen-binding fragments containing one or more individual germline mutations or combinations thereof, starting from the heavy chain variable region sequences and light chain variable region sequences disclosed herein. In certain embodiments, V H Domain and / or V L In other embodiments, all framework residues and / or CDR residues within the domain are mutated back to residues found in the original germline sequence from which the antibody originates. In other embodiments, only specific residues, for example, only mutant residues found within the first 8 amino acids of FR1, or only mutant residues found within CDR1, CDR2, or CDR3, are mutated back to the original germline sequence. In other embodiments, one or more framework residues and / or CDR residues are mutated to corresponding residues in a different germline sequence (i.e., a germline sequence different from the original germline sequence from which the antibody originates).

[0127] Furthermore, the antibodies of the present invention may contain any combination of two or more germline mutations within the framework and / or CDR region, for example, in which certain individual residues are mutated to corresponding residues in a particular germline sequence, while certain other residues different from the original germline sequence are maintained or mutated to corresponding residues in different germline sequences. Once obtained, antibodies and antigen-binding fragments containing one or more germline mutations can be readily tested for one or more desired properties, such as improved binding specificity, increased binding affinity, improved or enhanced (in some cases) biological properties of the antagonist or agonist, or decreased immunogenicity. Antibodies and antigen-binding fragments obtained in this general manner are encompassed by the present invention.

[0128] This specification provides anti-CD38 antibodies, anti-CD28 antibodies, or anti-CD38×anti-CD28 antibodies comprising a variant of any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein, having one or more conservative substitutions. For example, this disclosure includes anti-CD38 antibodies, anti-CD28 antibodies, or anti-CD38×anti-CD28 antibodies comprising an HCVR, LCVR, and / or CDR amino acid sequence having, for example, 10 or fewer, 8 or fewer, 6 or fewer, or 4 or fewer, 3 or fewer, 2 or 1, or other conservative amino acid substitutions to any of the HCVR, LCVR, and / or CDR amino acid sequences described in Tables 1, 4, or 7 of this specification.

[0129] The term "epitope" refers to an antigenic determinant that interacts with a specific antigen-binding site in the variable region of an antibody molecule, also known as a paratope. A single antigen may have two or more epitopes. Therefore, different antibodies may bind to different regions on an antigen and have different biological effects. Epitopes can be either steric or linear. Steric epitopes are generated by spatially juxtaposed amino acids from different segments of a linear polypeptide chain. Linear epitopes are those generated by adjacent amino acid residues within a polypeptide chain. In certain situations, epitopes may include sugar, phosphoryl, or sulfonyl groups on an antigen.

[0130] The terms “substantial identity” or “substantial identity” refer to nucleic acids or fragments thereof, indicating that, when optimally aligned with appropriate nucleotide insertions or deletions with another nucleic acid (or its complementary strand), they have nucleotide sequence identity in at least about 95%, more preferably at least about 96%, 97%, 98%, or 99% of the nucleotide bases, as measured by any well-known algorithm for sequence identity, such as FASTA, BLAST, or Gap, as described below. A nucleic acid molecule having substantial identity with a reference nucleic acid molecule may, in certain cases, encode a polypeptide having the same or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule.

[0131] When applied to polypeptides, the term “substantial similarity” or “substantially identical” means that two peptide sequences share at least 95% sequence identity, and more preferably at least 98% or 99%, when optimally aligned using default gap weights, such as by programmed GAP or BESTFIT. Preferably, non-identical residue positions are distinguished by different conserved amino acid substitutions. A “conservative amino acid substitution” is when an amino acid residue is replaced by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). Generally, conservative amino acid substitutions do not substantially alter the functional properties of the protein. If two or more amino acid sequences differ from each other by conservative substitutions, the percentage of sequence identity or degree of similarity may be adjusted upward to compensate for the conservative nature of the substitutions. Means for making this adjustment are well known to those skilled in the art. See, for example, Pearson (1994) Methods Mol. Biol. 24:307-331, incorporated herein by reference. Examples of amino acid groups having side chains with similar chemical properties include: (1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; (2) aliphatic-hydroxyl side chains: serine and threonine; (3) amide-containing side chains: asparagine and glutamine; (4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; (5) basic side chains: lysine, arginine, and histidine; (6) acidic side chains: aspartic acid and glutamic acid; and (7) sulfur-containing side chains: cysteine ​​and methionine. Preferred conserved amino acid substituents are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine. Alternatively, a conservative permutation is any change that has a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256:1443-1445, which is incorporated herein by reference. A "moderately conservative" permutation is any change that has a non-negative value in the PAM250 log-likelihood matrix.

[0132] Sequence similarity to polypeptides, also referred to as sequence identity, is typically measured using sequence analysis software. Protein analysis software matches similar sequences using similarity measures assigned to various substitutions, deletions, and other modifications, including conserved amino acid substitutions. For example, GCG software includes programs such as Gap and Bestfit, which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species, or between wild-type proteins and their mutant proteins. See, for example, GCG version 6.1. Polypeptide sequences can also be compared using FASTA, a program in GCG version 6.1, with default or recommended parameters. FASTA (e.g., FASTA2 and FASTA3) provides the best overlap region alignment and sequence identity percentage between the query sequence and the search sequence (Pearson (2000), above). Another preferred algorithm for comparing the sequences of the present invention with a database containing numerous sequences from different organisms is the computer program BLAST, particularly BLASTP or TBLASTN, using default parameters. For example, see Altschul et al. (1990) J.Mol.Biol.215:403-410 and Altschul et al. (1997) Nucleic Acids Res.25:3389-402, which are incorporated herein by reference.

[0133] germline mutation The anti-CD38 antibodies, anti-CD28 antibodies, and anti-CD38 / anti-CD28 bispecific antigen-binding molecules disclosed herein may include one or more amino acid substitutions, insertions, and / or deletions in the framework of the heavy chain variable domain and / or the CDR region compared to the corresponding germline sequence from which the antibody is derived.

[0134] Provided herein are antibodies derived from any of the amino acid sequences disclosed herein, and antigen-binding fragments thereof, wherein one or more frameworks and / or one or more amino acids within the CDR region are mutated to a corresponding residue in the germline sequence from which the antibody is derived, or to a corresponding residue in another human germline sequence, or to a conserved amino acid substitution of the corresponding germline residue (such sequence changes are collectively referred to herein as “germline mutations”), and have weak or undetectable binding to the CD38 antigen or CD28 antigen.

[0135] Antibody binding properties As used herein, in relation to an antibody, immunoglobulin, antibody-binding fragment, or Fc-containing protein binding to any of a given antigen, such as a cell surface protein or a fragment thereof, the term “binding” typically refers to an interaction or association between at least two entities or molecular structures, such as an antibody-antigen interaction.

[0136] For example, when binding affinity is determined by surface plasmon resonance (SPR) technique in a BIAcore instrument using, for example, an antigen as a ligand and an antibody, Ig, antibody-binding fragment, or Fc-containing protein as the analyte (or antiligand), it is typically about 10 -7 M or less, about 10 -8 M or less, about 10 -9 K below M D This corresponds to the value. Cell-based binding strategies such as fluorescently labeled cell sorting (FACS) binding assays are also routinely used, and FACS data correlate well with other methods such as radioligand competitive binding and SPR (Benedict, CA, J Immunol Methods. 1997, 201(2):223-31; Geuijen, CA, et al. J Immunol Methods. 2005, 302(1-2):68-77).

[0137] Therefore, the antibodies or antigen-binding proteins disclosed herein have a K2-10-fold lower affinity than their affinity for binding to nonspecific antigens (e.g., BSA, casein). DIt binds to a predetermined antigen or cell surface molecule (receptor) having affinity corresponding to the value. According to this disclosure, K is 10 times or less than that of a nonspecific antigen. D The antibody affinity corresponding to the value may be considered undetectable binding, but such an antibody can be paired with a second antigen-binding arm for the production of the bispecific antibody of the present invention.

[0138] "K D The term (M) refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, or the dissociation equilibrium constant of an antibody-binding fragment that binds to an antibody or antigen. D There is an inverse relationship between and binding affinity, and therefore, K D The smaller the value, the higher the affinity, i.e., the stronger it is. Therefore, the terms "higher affinity" or "stronger affinity" refer to a higher ability to form an interaction, and thus a smaller K. D Regarding the values, conversely, the terms "lower affinity" or "weaker affinity" indicate a lower ability to form an interaction, and therefore a higher K. D Regarding the value. In some situations, the binding affinity (or K) of a particular molecule (e.g., antibody) to its interaction partner molecule (e.g., antigen X) is the value. D If the binding affinity of that molecule (e.g., antibody) is higher compared to the binding affinity of another interaction partner molecule (e.g., antigen Y), then a larger K D The value (lower or weaker affinity) is smaller K D It can be expressed as a binding ratio determined by dividing by a value (higher or stronger affinity), and in some cases, it can be expressed as a binding affinity that is 5 or 10 times higher.

[0139] "k d The term (sec-1 or 1 / s) refers to the dissociation rate constant of a particular antibody-antigen interaction, or the dissociation rate constant of an antibody or antibody-binding fragment. This value is k off It is also called a value.

[0140] "k aThe term "(M-1×sec-1 or 1 / M / s)" refers to the association rate constant of a specific antibody-antigen interaction, or the association rate constant of an antibody or an antibody binding fragment.

[0141] "K A "(M-1 or 1 / M) refers to the association equilibrium constant of a specific antibody-antigen interaction, or the association equilibrium constant of an antibody or an antibody binding fragment. The association equilibrium constant is obtained by dividing k a by k d .

[0142] The term "EC50" or "EC 50 " refers to the half maximal effective concentration, including the concentration of an antibody that induces a response midway between the baseline and the maximum after a specified exposure time. EC 50 basically represents the concentration of an antibody at which 50% of its maximum effect is observed. In certain embodiments, the EC 50 value is equal to, for example, the concentration of an antibody of the invention that gives half of the maximum binding to cells expressing CD28 or a tumor-associated antigen (e.g., CD38), determined by, for example, a FACS binding assay. Thus, an increase in EC 50 or the half maximal effective concentration value is observed to result in a decrease or attenuation of binding.

[0143] In one embodiment, the decrease in binding can be defined as an increase in the EC 50 antibody concentration that enables half of the maximum amount of binding to target cells.

[0144] In another embodiment, the EC 50 value represents the concentration of an antibody of the invention that induces maximum half depletion of target cells by the cytotoxic activity of T cells. Thus, an increase in cytotoxic activity (e.g., T cell-mediated tumor cell killing) is observed with a decrease in EC 50 , or the half maximal effective concentration value.

[0145] Bispecific antigen-binding molecule The antibodies of the present invention may be monospecific, bispecific, or multispecific. Multispecific antibodies may be specific to different epitopes of one target polypeptide, or may contain antigen-binding domains specific to multiple target polypeptides. See, for example, Tutt et al., 1991, J.Immunol. 147:60-69 and Kufer et al., 2004, Trends Biotechnol. 22:238-244. The anti-CD38 monospecific antibody, anti-CD28 monospecific antibody, or anti-CD38 × anti-CD28 bispecific antibody of this disclosure may be ligated to another functional molecule, such as another peptide or protein, or may be co-expressed with another functional molecule, such as another peptide or protein. For example, an antibody or a fragment thereof can be functionally linked (e.g., by chemical bonding, gene fusion, non-covalent association, or other means) to one or more other molecular entities, such as another antibody or antibody fragment, to produce a bispecific or polyspecific antibody having a second or additional binding specificity.

[0146] The use of the terms “anti-CD28 antibody” or “anti-CD38 antibody” herein is intended to include both monospecific anti-CD28 antibodies or anti-CD38 antibodies, as well as bispecific antibodies comprising a CD28-binding arm and a CD38-binding arm. Accordingly, this disclosure includes bispecific antibodies in which one arm of the immunoglobulin binds to human CD28 and the other arm of the immunoglobulin is specific to human CD38. The CD28-binding arm may comprise either the HCVR / LCVR or CDR amino acid sequence listed in Table 4 herein.

[0147] In certain embodiments, the CD28-binding arm binds to human CD28 and promotes human T cell activation. In certain embodiments, the CD28-binding arm binds to human CD28 and induces human T cell activation. In other embodiments, the CD28-binding arm binds to human CD28 and, in association with a bispecific or multispecific antibody, induces tumor-associated antigen-expressing cell toxicity. The CD38-binding arm may contain any of the HCVR / LCVR or CDR amino acid sequences listed in Table 1 of this specification.

[0148] According to certain exemplary embodiments, the present invention includes bispecific antigen-binding molecules that specifically bind to CD28 and CD38. Such molecules may be referred to herein, for example, as “anti-CD38 × anti-CD28,” or “anti-CD38 / anti-CD28,” or “anti-CD38 × CD28” or “CD38 × CD28” bispecific molecules, or other similar terms (e.g., anti-CD28 / anti-CD38).

[0149] As used herein, the term "CD38" refers to the human CD38 protein unless otherwise specified that it is derived from a non-human species (e.g., "mouse CD38," "monkey CD38," etc.). The human CD38 protein has the amino acid sequence shown in SEQ ID NO: 62 (Human CD38 Extracellular Domain (V43-I300).mFc) and / or the amino acid sequence described in NCBI accession numbers NP_001766.2 or NM_001775.3.

[0150] Human CD38 extracellular domain (V43-I300).mFc (immunogen) amino acid [ka] The bispecific antigen-binding molecule that specifically binds to CD38 and CD28 has a K+ of approximately 200 μM when measured by in vitro affinity binding assay. D It may also contain an anti-CD28 antigen-binding molecule that binds to CD28 with weak binding affinity, such as exhibiting the following characteristics.

[0151] As used herein, the expression “antigen-binding molecule” means a protein, polypeptide, or molecular complex comprising, or consisting of, at least one complementarity-determining region (CDR) that binds specifically to a particular antigen, either alone or in combination with one or more additional CDRs and / or framework regions (FRs). In certain embodiments, the antigen-binding molecule is an antibody or a fragment of an antibody, the terms of which are defined elsewhere herein.

[0152] As used herein, the expression “bispecific antigen-binding molecule” means a protein, polypeptide, or molecular complex comprising at least a first antigen-binding domain and a second antigen-binding domain. Each antigen-binding domain within a bispecific antigen-binding molecule comprises at least one CDR, either alone or in combination with one or more additional CDRs and / or FRs, which specifically bind to a particular antigen. In the context of the present invention, the first antigen-binding domain specifically binds to a first antigen (e.g., CD38), and the second antigen-binding domain specifically binds to a second distinct antigen (e.g., CD28).

[0153] In certain exemplary embodiments of the present invention, the bispecific antigen-binding molecule is a bispecific antibody. Each antigen-binding domain of the bispecific antibody comprises a heavy chain variable domain (HCVR) and a light chain variable domain (LCVR). In the context of a bispecific antigen-binding molecule (e.g., a bispecific antibody) comprising first and second antigen-binding domains, the CDR of the first antigen-binding domain may be denoted by the prefix "D1", and the CDR of the second antigen-binding domain may be denoted by the prefix "D2". Thus, the CDRs of the first antigen-binding domain may be referred to herein as D1-HCDR1, D1-HCDR2, and D1-HCDR3, and the CDRs of the second antigen-binding domain may be referred to herein as D2-HCDR1, D2-HCDR2, and D2-HCDR3.

[0154] In certain exemplary embodiments, the isolated bispecific antigen-binding molecule comprises a first antigen-binding domain comprising (a) three heavy chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) contained within a heavy chain variable region (HCVR) containing the amino acid sequence of SEQ ID NO: 2, and (b) three light chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3) contained within a light chain variable region (LCVR) containing the amino acid sequence of SEQ ID NO: 18. In some cases, the isolated bispecific antigen-binding molecule comprises a first antigen-binding domain comprising HCDR1 containing the amino acid sequence of SEQ ID NO: 4, HCDR2 containing the amino acid sequence of SEQ ID NO: 6, and HCDR3 containing the amino acid sequence of SEQ ID NO: 8. In some cases, the isolated bispecific antigen-binding molecule comprises a first antigen-binding domain comprising LCDR1 containing the amino acid sequence of SEQ ID NO: 20, LCDR2 containing the amino acid sequence of SEQ ID NO: 22, and LCDR3 containing the amino acid sequence of SEQ ID NO: 24. In some cases, the first antigen-binding domain includes HCVR containing the amino acid sequence of SEQ ID NO: 2 and LCVR containing the amino acid sequence of SEQ ID NO: 18.

[0155] In certain exemplary embodiments, the isolated bispecific antigen-binding molecule includes a second antigen-binding domain comprising (a) three heavy chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) contained within a heavy chain variable region (HCVR) containing the amino acid sequence of SEQ ID NO: 10, and (b) three light chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3) contained within a light chain variable region (LCVR) containing the amino acid sequence of SEQ ID NO: 18. The second antigen-binding domain may include HCDR1 containing the amino acid sequence of SEQ ID NO: 12, HCDR2 containing the amino acid sequence of SEQ ID NO: 14, and HCDR3 containing the amino acid sequence of SEQ ID NO: 16. The second antigen-binding domain may also include LCDR1 containing the amino acid sequence of SEQ ID NO: 20, LCDR2 containing the amino acid sequence of SEQ ID NO: 22, and LCDR3 containing the amino acid sequence of SEQ ID NO: 24. The second antigen-binding domain may also include HCVR containing the amino acid sequence of SEQ ID NO: 10, and LCVR containing the amino acid sequence of SEQ ID NO: 18.

[0156] In certain exemplary embodiments, the isolated bispecific antigen-binding molecule comprises a first antigen-binding domain comprising (a) three heavy chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) contained within a heavy chain variable region (HCVR) containing the amino acid sequence of SEQ ID NO: 32, and (b) three light chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3) contained within a light chain variable region (LCVR) containing the amino acid sequence of SEQ ID NO: 48. In some cases, the isolated bispecific antigen-binding molecule comprises HCDR1 containing the amino acid sequence of SEQ ID NO: 34, HCDR2 containing the amino acid sequence of SEQ ID NO: 36, and HCDR3 containing the amino acid sequence of SEQ ID NO: 38. In some cases, the isolated bispecific antigen-binding molecule comprises LCDR1 containing the amino acid sequence of SEQ ID NO: 50, LCDR2 containing the amino acid sequence of SEQ ID NO: 52, and LCDR3 containing the amino acid sequence of SEQ ID NO: 54. In some cases, the first antigen-binding domain comprises HCVR containing the amino acid sequence of SEQ ID NO: 32, and LCVR containing the amino acid sequence of SEQ ID NO: 48.

[0157] In certain exemplary embodiments, the isolated bispecific antigen-binding molecule includes a second antigen-binding domain comprising (a) three heavy chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) contained within a heavy chain variable region (HCVR) containing the amino acid sequence of SEQ ID NO: 40, and (b) three light chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3) contained within a light chain variable region (LCVR) containing the amino acid sequence of SEQ ID NO: 48. The second antigen-binding domain may include HCDR1 containing the amino acid sequence of SEQ ID NO: 42, HCDR2 containing the amino acid sequence of SEQ ID NO: 44, and HCDR3 containing the amino acid sequence of SEQ ID NO: 46. The second antigen-binding domain may also include LCDR1 containing the amino acid sequence of SEQ ID NO: 50, LCDR2 containing the amino acid sequence of SEQ ID NO: 52, and LCDR3 containing the amino acid sequence of SEQ ID NO: 54. The second antigen-binding domain may also include HCVR containing the amino acid sequence of SEQ ID NO: 40, and LCVR containing the amino acid sequence of SEQ ID NO: 48.

[0158] In certain exemplary embodiments, the isolated bispecific antigen-binding molecule comprises (a) a first antigen-binding domain comprising the HCDR1 amino acid sequence of SEQ ID NO: 4, the HCDR2 amino acid sequence of SEQ ID NO: 6, and the HCDR3 amino acid sequence of SEQ ID NO: 8, and (b) a second antigen-binding domain comprising the HCDR1 amino acid sequence of SEQ ID NO: 12, the HCDR2 amino acid sequence of SEQ ID NO: 14, and the HCDR3 amino acid sequence of SEQ ID NO: 16. In some cases, the isolated bispecific antigen-binding molecule comprises the LCDR1 amino acid sequence of SEQ ID NO: 20, the LCDR2 amino acid sequence of SEQ ID NO: 22, and the LCDR3 amino acid sequence of SEQ ID NO: 24.

[0159] In certain exemplary embodiments, the isolated bispecific antigen-binding molecule comprises (a) a first antigen-binding domain comprising the HCDR1 amino acid sequence of SEQ ID NO: 34, the HCDR2 amino acid sequence of SEQ ID NO: 36, and the HCDR3 amino acid sequence of SEQ ID NO: 38, and (b) a second antigen-binding domain comprising HCDR1 comprising the amino acid sequence of SEQ ID NO: 42, HCDR2 comprising the amino acid sequence of SEQ ID NO: 44, and HCDR3 comprising the amino acid sequence of SEQ ID NO: 46. In some cases, the isolated bispecific antigen-binding molecule comprises the LCDR1 amino acid sequence of SEQ ID NO: 50, the LCDR2 amino acid sequence of SEQ ID NO: 52, and the LCDR3 amino acid sequence of SEQ ID NO: 54.

[0160] In certain exemplary embodiments, the isolated bispecific antigen-binding molecule comprises (a) a first antigen-binding domain comprising HCDR1 comprising the amino acid sequence of SEQ ID NO: 4, HCDR2 comprising the amino acid sequence of SEQ ID NO: 6, and HCDR3 comprising the amino acid sequence of SEQ ID NO: 8; LCDR1 comprising the amino acid sequence of SEQ ID NO: 20, LCDR2 comprising the amino acid sequence of SEQ ID NO: 22, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 24; and (b) a second antigen-binding domain comprising HCDR1 comprising the amino acid sequence of SEQ ID NO: 12, HCDR2 comprising the amino acid sequence of SEQ ID NO: 14, and HCDR3 comprising the amino acid sequence of SEQ ID NO: 16; the LCDR1 amino acid sequence of SEQ ID NO: 20, the LCDR2 amino acid sequence of SEQ ID NO: 22, and the LCDR3 amino acid sequence of SEQ ID NO: 24. In some cases, the isolated bispecific antigen-binding molecule comprises (a) a first antigen-binding domain comprising the HCVR amino acid sequence of SEQ ID NO: 2 and the LCVR amino acid sequence of SEQ ID NO: 18; and (b) a second antigen-binding domain comprising the HCVR amino acid sequence of SEQ ID NO: 10 and the LCVR amino acid sequence of SEQ ID NO: 18.

[0161] In certain exemplary embodiments, the isolated bispecific antigen-binding molecule comprises (a) a first antigen-binding domain comprising an HCVR CDR containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 and 32, and an LCVR CDR containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 and 48, and (b) a second antigen-binding domain that specifically binds to human CD28. Optionally, the first antigen-binding domain comprises a CDR derived from an HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 2 / 18 and 32 / 48. Optionally, the first antigen-binding domain comprises HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domains selected from the group consisting of SEQ ID NOs: 4-6-8-20-22-24 and 34-36-38-50-52-54, respectively. Optionally, the first antigen-binding domain comprises an HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 2 / 18 and 32 / 48. In some cases, the second antigen-binding domain contains an HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 10 / 18 and 40 / 48.

[0162] In certain exemplary embodiments, isolated bispecific antigen-binding molecules compete for binding to CD38 or bind to the same epitope on CD38 as a reference antibody, the reference antibody comprising an antibody or its antigen-binding fragment, or a bispecific anti-CD38 / CD28 antibody disclosed herein.

[0163] In certain exemplary embodiments, isolated bispecific antigen-binding molecules compete for binding to human CD3 or bind to the same epitope on human CD3 as a reference antibody, the reference antibody comprising an antibody or its antigen-binding fragment, or a bispecific anti-CD38 / CD28 antibody disclosed herein.

[0164] The bispecific antigen-binding molecules described above or discussed herein may be bispecific antibodies. In some cases, the bispecific antibody contains a human IgG heavy chain constant region. In some cases, the human IgG heavy chain constant region is isotype IgG1. In some cases, the human IgG heavy chain constant region is isotype IgG4. In various embodiments, the bispecific antibody, compared to a wild-type hinge of the same isotype,

number

[0165] The first antigen-binding domain and the second antigen-binding domain can be directly or indirectly linked to each other to form the bispecific antigen-binding molecule of the present invention. Alternatively, the first antigen-binding domain and the second antigen-binding domain may each be linked to a separate multimerizing domain. The association of one multimerizing domain with another facilitates the association between the two antigen-binding domains, thereby forming the bispecific antigen-binding molecule. As used herein, “multimerizing domain” is any macromolecule, protein, polypeptide, peptide, or amino acid having the ability to associate with a second multimerizing domain of the same or similar structure or configuration. For example, the multimerizing domain may be immunoglobulin C H The polypeptide may also contain three domains. Non-limiting examples of polymer-forming components include (C H 2-C H The Fc portion of immunoglobulins (including 3 domains), for example, the Fc domain of IgG selected from isotypes IgG1, IgG2, IgG3, and IgG4, as well as any allotype within each isotype group.

[0166] The bispecific antigen-binding molecule of the present invention will typically contain two multimerizing domains, for example, two Fc domains, each being an individual part of a separate antibody heavy chain. The first and second multimerizing domains may be of the same IgG isotype, for example, IgG1 / IgG1, IgG2 / IgG2, IgG4 / IgG4. Alternatively, the first and second multimerizing domains may be of different IgG isotypes, for example, IgG1 / IgG2, IgG1 / IgG4, IgG2 / IgG4.

[0167] In certain embodiments, the multimerizing domain is an Fc fragment or an amino acid sequence of 1 to about 200 amino acids in length containing at least one cysteine ​​residue. In other embodiments, the multimerizing domain is a cysteine ​​residue or a short-chain cysteine-containing peptide. Other multimerizing domains include peptides or polypeptides containing or comprising a leucine zipper, a helix-loop motif, or a coiled-coil motif.

[0168] Any bispecific antibody format or technique may be used to produce the bispecific antigen-binding molecules of the present invention. For example, an antibody or fragment thereof having a first antigen-binding specificity can be functionally linked (e.g., by chemical linkage, gene fusion, non-covalent association, or other means) to one or more other molecular entities, such as another antibody or antibody fragment having a second binding specificity, to produce a bispecific antigen-binding molecule. Specific examples of bispecific formats that may be used in the context of the present invention include, but are not limited to, scFv-based or diabody bispecific formats, IgG-scFv fusions, dual variable region (DVD)-Ig, quadroma, knob-into-hole, common light chain (e.g., a common light chain with a knob-into-hole), CrossMab, CrossFab, (SEED) body, leucine zipper, duobody, IgG1 / IgG2, dual-acting Fab (DAF)-IgG, and Mab 2One example is the bispecific format (see, for example, Klein et al. 2012, mAbs 4:6, 1-11, and the references cited therein for a discussion of the aforementioned format).

[0169] In the context of the bispecific antigen-binding molecules of the present invention, the multimerizing domain, for example, the Fc domain, may include one or more amino acid modifications (e.g., insertions, deletions, or substitutions) compared to the naturally occurring version of the wild-type Fc domain. For example, the present invention includes bispecific antigen-binding molecules that include one or more modifications in the Fc domain, resulting in a modified Fc domain having an altered binding interaction (e.g., enhanced or reduced) between Fc and FcRn. In one embodiment, the bispecific antigen-binding molecule is C H 2 regions or C H The modification includes alterations in three regions, which increase the affinity of the Fc domain to FcRn in an acidic environment (e.g., endosomes with a pH in the range of approximately 5.5 to 6.0). Non-limiting examples of such Fc alterations include, for example, alterations at position 250 (e.g., E or Q), position 250 and position 428 (e.g., L or F), position 252 (e.g., L / Y / F / W or T), position 254 (e.g., S or T), and position 256 (e.g., S / R / Q / E / D or T), or alterations at position 428 and / or 433 (e.g., L / R / S / P / Q or K) and / or position 434 (e.g., H / F or Y), or alterations at position 250 and / or 428, or alterations at position 307 or 308 (e.g., 308F, V308F), and position 434. In one embodiment, modifications include the 428L (e.g., M428L) and 434S (e.g., N434S) modifications, the 428L, 259I (e.g., V259I), and 308F (e.g., V308F) modifications, the 433K (e.g., H433K) and 434 (e.g., 434Y) modifications, the 252, 254, and 256 (e.g., 252Y, 254T, and 256E) modifications, the 250Q and 428L modifications (e.g., T250Q and M428L), and the 307 and / or 308 modifications (e.g., 308F and / or 308P).

[0170] This disclosure also relates to the first Ig C H 3 domains and 2 Ig C H It contains a bispecific antigen-binding molecule with three domains, and the first and second IgC H The three domains differ from each other at least at one amino acid, and this difference at at least one amino acid reduces the binding of the bispecific antibody to protein A compared to a bispecific antibody lacking amino acid differences. In one embodiment, the first Ig C H The 3 domains bind to protein A, and the second Ig C H The 3 domains contain mutations that reduce or eliminate protein A binding, such as the H95R modification (according to the IMGT exon numbering rules; H435R according to the EU numbering rules). Second C H 3 may further include the Y96F modification (according to IMGT, Y436F according to the EU). See, for example, U.S. Patent No. 8,586,713. Second C H Further modifications that may be found within 3 include: for IgG1 antibodies, D16E, L18M, N44S, K52N, V57M, and V82I (according to IMGT; D356E, L358M, N384S, K392N, V397M, and V422I in the EU); for IgG2 antibodies, N44S, K52N, and V82I (according to IMGT; N384S, K392N, and V422I in the EU); and for IgG4 antibodies, Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I (according to IMGT; Q355R, N384S, K392N, V397M, R409K, E419Q, and V422I in the EU).

[0171] In certain embodiments, the Fc domain may be a chimeric combination of Fc sequences derived from two or more immunoglobulin isotypes. For example, the chimeric Fc domain may be human IgG1, human IgG2, or human IgG4 C H C originating from 2 regions H Part or all of the 2 sequences, and C derived from human IgG1, human IgG2, or human IgG4. H3. It may contain part or all of the sequences. The chimeric Fc domain may also contain a chimeric hinge region. For example, the chimeric hinge may contain an "upper hinge" sequence derived from the human IgG1, human IgG2, or human IgG4 hinge region combined with a "lower hinge" sequence derived from the human IgG1, human IgG2, or human IgG4 hinge region. A specific example of a chimeric Fc domain that may be contained in any of the antigen-binding molecules described herein is [IgG4 C] from the N-terminus to the C-terminus. H It includes [1]-[IgG4 upper hinge]-[IgG2 lower hinge]-[IgG4 CH2]-[IgG4 CH3]. Another example of a chimeric Fc domain that may be included in any of the antigen-binding molecules described herein is [IgG1 C] from the N-terminus to the C-terminus. H This includes [1]-[IgG1 upper hinge]-[IgG2 lower hinge]-[IgG4 CH2]-[IgG1 CH3]. These and other examples of chimeric Fc domains that may be included in any of the antigen-binding molecules of the present invention are described in U.S. Patent Application Publication No. 2014 / 0243504, published on 28 August 2014, which is incorporated herein by reference in its entirety. Chimeric Fc domains and their variants having these common structural arrangements may have altered Fc receptor binding, thereby affecting Fc effector function.

[0172] Array variant The antibodies and bispecific antigen-binding molecules of the present invention may include one or more amino acid substitutions, insertions, and / or deletions in the framework and / or CDR region of the heavy and light chain variable domains compared to the corresponding germline sequences from which the individual antigen-binding domains are derived. Such mutations can be readily identified by comparing the amino acid sequences disclosed herein with germline sequences available, for example, from publicly available antibody sequence databases. The antigen-binding molecules of the present invention may include antigen-binding domains derived from any of the amino acid sequences disclosed herein, in which one or more amino acids in one or more frameworks and / or CDR regions are mutated to corresponding residues in the germline sequence from which the antibody is derived, or to corresponding residues in another human germline sequence, or to conserved amino acid substitutions of the corresponding germline residues (such sequence changes are collectively referred to herein as “germline mutations”). Those skilled in the art can readily produce many antibodies and antigen-binding fragments containing one or more individual germline mutations or combinations thereof, starting from the heavy chain variable region sequences and light chain variable region sequences disclosed herein. In certain embodiments, V H Domain and / or V L In other embodiments, all framework residues and / or CDR residues within the domain are mutated back to residues found in the original germline sequence from which the antigen-binding domain originally originated. In other embodiments, only specific residues, for example, only mutant residues found within the first 8 amino acids of FR1, or only mutant residues found within CDR1, CDR2, or CDR3, are mutated back to the original germline sequence. In other embodiments, one or more framework residues and / or CDR residues are mutated to corresponding residues in a different germline sequence (i.e., a germline sequence different from the germline sequence from which the antigen-binding domain originally originated).

[0173] Furthermore, the antigen-binding domain may contain any combination of two or more germline mutations within the framework and / or CDR region, for example, in which specific individual residues are mutated to corresponding residues in a particular germline sequence, while certain other residues different from the original germline sequence are maintained or mutated to corresponding residues in a different germline sequence. Once obtained, an antigen-binding domain containing one or more germline mutations can be readily tested for one or more desired properties, such as improved binding specificity, increased binding affinity, improved or enhanced (in some cases) biological properties of the antagonist or agonist, or decreased immunogenicity. A bispecific antigen-binding molecule containing one or more antigen-binding domains obtained in this general manner is encompassed in this disclosure.

[0174] pH dependent binding The present invention comprises anti-CD38 antibodies, anti-CD28 antibodies, and anti-CD38 × anti-CD28 bispecific antigen-binding molecules having pH-dependent binding properties. For example, the anti-CD38 antibody of the present invention may show decreased binding to CD38 at acidic pH compared to neutral pH. Alternatively, the anti-CD38 antibody of the present invention may show enhanced binding to CD38 at acidic pH compared to neutral pH. The term "acidic pH" includes pH values ​​less than about 6.2, for example, about 6.0, 5.95, 5.9, 5.85, 5.8, 5.75, 5.7, 5.65, 5.6, 5.55, 5.5, 5.45, 5.4, 5.35, 5.3, 5.25, 5.2, 5.15, 5.1, 5.05, and 5.0. As used herein, the term "neutral pH" means a pH of about 7.0 to about 7.4. The term "neutral pH" includes pH values ​​of approximately 7.0, 7.05, 7.1, 7.15, 7.2, 7.25, 7.3, 7.35, and 7.4.

[0175] In a specific example, "a decrease in binding to the antigen at acidic pH compared to neutral pH" indicates that the K of the antibody that binds to the antigen at acidic pH is reduced. D The value and the K of the antibody that binds to the antigen at a neutral pH. D It is expressed as a ratio to (or vice versa) the value. For example, an antibody or its antigen-binding fragment is expressed as an acidic / neutral potassium ratio of approximately 3.0 or higher.D When a ratio is observed, for the purposes of the invention, it may be considered to exhibit "a decrease in binding to CD38 at acidic pH compared to neutral pH." In certain exemplary embodiments, the acidic / neutral K ratio to the antibody or antigen-binding fragment of the present invention D The ratios can be approximately 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 20.0, 25.0, 30.0, 40.0, 50.0, 60.0, 70.0, and 100.0 or higher.

[0176] Antibodies with pH-dependent binding properties can be obtained, for example, by screening a population of antibodies for decreased (or increased) binding to a specific antigen at acidic pH compared to neutral pH. In addition, modification of the antigen-binding domain at amino acid concentration can produce antibodies with pH-dependent properties. For example, by substituting one or more amino acids in the antigen-binding domain (e.g., within the CDR) with histidine residues, antibodies with decreased antigen binding at acidic pH compared to neutral pH can be obtained.

[0177] Antibodies containing Fc variants According to certain embodiments of the present invention, for example, anti-CD38 antibodies, anti-CD28 antibodies, and anti-CD38 × anti-CD28 bispecific antigen-binding molecules are provided, comprising an Fc domain containing one or more mutations that enhance or reduce antibody binding to the FcRn receptor at acidic pH compared to neutral pH. For example, the present invention provides an Fc domain containing C H 2 or C HAn antibody comprising a mutation in the 3 regions, the mutation increasing the affinity of the Fc domain for FcRn in an acidic environment (e.g., in an endosome with a pH in the range of about 5.5 to about 6.0). Such a mutation can result in an increase in the serum half-life of the antibody when administered to an animal. Non-limiting examples of such Fc modifications include, for example, modifications at position 250 (e.g., E or Q), positions 250 and 428 (e.g., L or F), position 252 (e.g., L / Y / F / W or T), position 254 (e.g., S or T), and position 256 (e.g., S / R / Q / E / D or T), or modifications at position 428 and / or 433 (e.g., H / L / R / S / P / Q or K) and / or position 434 (e.g., H / F or Y), or modifications at position 250 and / or 428, or modifications at position 307 or 308 (e.g., 308F, V308F), and position 434. In one embodiment, the modifications include 428L (e.g., M428L) and 434S (e.g., N434S) modifications, 428L, 259I (e.g., V259I), and 308F (e.g., V308F) modifications, 433K (e.g., H433K) and 434 (e.g., 434Y) modifications, 252, 254, and 256 (e.g., 252Y, 254T, and 256E) modifications, 250Q and 428L modifications (e.g., T250Q and M428L), and 307 and / or 308 modifications (e.g., 308F and / or 308P).

[0178] For example, the present disclosure includes an anti-CD38 antibody, an anti-CD28 antibody, an anti-CD38×anti-CD28 bispecific antigen-binding molecule comprising an Fc domain comprising one or more pairs or groups of mutations selected from the group consisting of: 250Q and 248L (e.g., T250Q and M248L); 252Y, 254T and 256E (e.g., M252Y, S254T and T256E); 428L and 434S (e.g., M428L and N434S); and 433K and 434F (e.g., H433K and N434F). All possible combinations of the foregoing Fc domain mutations and other mutations within the antibody variable domains disclosed herein are contemplated to be within the scope of the present invention.

[0179] Biological Characteristics of Antibodies and Bispecific Antigen-Binding Molecules The present invention includes antibodies and antigen-binding fragments thereof that bind to human CD38 and / or CD28 with high affinity (e.g., K values in the nanomolar or sub-nanomolar range). D value).

[0180] According to certain embodiments, the present invention includes antibodies, antigen-binding fragments of antibodies, and bispecific antibodies that bind to human CD38 with a K (e.g., at 25°C) of less than about 10 nM as measured by surface plasmon resonance using, for example, the assay format defined in Example 5 herein. In certain embodiments, the antibody or antigen-binding fragment of the present invention has a K of less than about 20 nM, less than about 10 nM, less than about 8 nM, less than about 7 nM, less than about 6 nM, less than about 5 nM, less than about 4 nM, less than about 3 nM, less than about 2 nM, less than about 1 nM, less than about 800 pM, less than about 700 pM, less than about 500 pM, less than about 400 pM, less than about 300 pM, less than about 200 pM, less than about 100 pM, less than about 50 pM, or less than about 25 pM for binding to CD38 as measured by surface plasmon resonance using, for example, the assay format defined in Example 5 herein or an assay substantially similar thereto. D to bind to CD38.

[0181] According to certain embodiments, the present invention includes antibodies, antigen-binding fragments of antibodies, and bispecific antibodies that bind to human CD38 with a K of less than about 26 nM as measured by surface plasmon resonance using, for example, the assay format defined in Example 5 herein. D ​​The present invention comprises an antibody that binds to human CD28 at (for example, 25°C), an antigen-binding fragment of the antibody, and a bispecific antibody. In certain embodiments, the antibody or antigen-binding fragment of the present invention, when measured by surface plasmon resonance using, for example, the assay format defined in Example 5 of this specification, or a substantially similar assay, has a K content of less than about 20 nM, less than about 10 nM, less than about 8 nM, less than about 7 nM, less than about 6 nM, less than about 5 nM, less than about 4 nM, less than about 3 nM, less than about 2 nM, less than about 1 nM, less than about 800 pM, less than about 700 pM, less than about 500 pM, less than about 400 pM, less than about 300 pM, less than about 200 pM, less than about 100 pM, less than about 50 pM, or less than about 25 pM. D The antibody binds to CD28. The Disclosure also includes antibodies and antigen-binding fragments thereof that bind to CD38 with a dissociation half-life (t1 / 2) of more than about 8 minutes or more than 15 minutes when measured by surface plasmon resonance at 25°C using, for example, the assay format defined in Example 5 of the Specified Specification, or a substantially similar assay. In certain embodiments, the antibody or antigen-binding fragment of the Disclosure binds to CD38 with a t1 / 2 of more than about 3 minutes, more than about 4 minutes, more than about 10 minutes, more than about 20 minutes, more than about 30 minutes, more than about 40 minutes, more than about 50 minutes, more than about 60 minutes, more than about 70 minutes, more than about 80 minutes, more than about 90 minutes, more than about 100 minutes, more than about 110 minutes, or more than about 120 minutes when measured by surface plasmon resonance at 25°C using, for example, the assay format defined in Example 5 of the Specified Specification, or a substantially similar assay. The present invention relates to a bispecific antigen-binding molecule (e.g., a bispecific antibody) that, when measured by surface plasmon resonance at 25°C, binds to CD38 in more than 10 minutes, for a period of time greater than approximately 10 minutes, using, for example, the assay format defined in Example 5 of this specification or a substantially similar assay.

[0182] The Disclosure also includes antibodies and antigen-binding fragments thereof that bind to CD28 with a dissociation half-life (t1 / 2) of more than about 5 minutes or more than about 18 minutes when measured by surface plasmon resonance at 25°C using, for example, the assay format defined in Example 5 of the Specified Specification, or a substantially similar assay. In certain embodiments, the antibodies or antigen-binding fragments of the Disclosure bind to CD28 with a t1 / 2 of more than about 3 minutes, more than about 4 minutes, more than about 10 minutes, more than about 20 minutes, more than about 30 minutes, more than about 40 minutes, more than about 50 minutes, more than about 60 minutes, more than about 70 minutes, more than about 80 minutes, more than about 90 minutes, more than about 100 minutes, more than about 110 minutes, or more than about 120 minutes when measured by surface plasmon resonance at 25°C using, for example, the assay format defined in Example 5 of the Specified Specification, or a substantially similar assay. The present invention relates to a bispecific antigen-binding molecule (e.g., a bispecific antibody) that, when measured by surface plasmon resonance at 25°C, binds to CD38 in more than 10 minutes, for a period of time greater than approximately 10 minutes, using, for example, the assay format defined in Example 5 of this specification or a substantially similar assay.

[0183] The disclosure also includes antibodies and antigen-binding fragments thereof that specifically bind to human cell lines expressing endogenous CD38 (e.g., NCI-H929, MOLP-8, or WSU-DLCL2 tumor cells) as determined by in vivo xenotumor studies or substantially similar assays described in Examples 9-14.

[0184] The disclosure also includes anti-CD38 × anti-CD28 bispecific antigen-binding molecules exhibiting one or more characteristics selected from the group consisting of: (a) inhibiting tumor growth in immunodeficient mice having a human multiple myeloma xenograft; (b) suppressing tumor growth of engrafted tumors in immunodeficient mice having a human multiple myeloma xenograft (see, for example, Examples 9-14); and (c) suppressing tumor growth of syngeneic melanoma cells engineered to express human CD38 in immunocompetent mice.

[0185] This disclosure includes a bispecific antigen-binding molecule (e.g., a bispecific antibody) that can simultaneously bind to human CD38 and human CD28. The extent to which the bispecific antigen-binding molecule binds to cells expressing CD38 and / or CD28 can be evaluated by fluorescent cell sorting (FACS).

[0186] For example, the present invention includes an antibody, an antigen-binding fragment, and a bispecific antibody thereof that specifically binds to a human T cell line expressing CD38 but not CD28, and / or BCMA-expressing cells.

[0187] This disclosure includes antibodies, antigen-binding fragments, and bispecific antibodies thereof that bind to human CD38 and / or CD28 and induce T cell activation.

[0188] The present invention includes an anti-CD38 × anti-CD28 bispecific antigen-binding molecule that can deplete or reduce tumor antigen-expressing cells in a subject (see, for example, Examples 9-12 or substantially similar assays). For example, according to certain embodiments, an anti-CD38 × anti-CD28 bispecific antigen-binding molecule is provided, and a single or multiple administration of 0.04 mg / kg, 0.4 mg / kg, or 4 mg / kg of the bispecific antigen-binding molecule to a subject causes a reduction in the number of CD38-expressing cells in the subject (e.g., tumor growth in the subject is suppressed or inhibited).

[0189] Epitope mapping and related technologies In this specification, the epitopes on CD38 and / or CD28 to which antigen-binding molecules bind may consist of a single continuous sequence of three or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) amino acids of the CD38 or CD28 protein. Alternatively, the epitope may consist of a plurality of discontinuous amino acids (or amino acid sequences) of CD38 or CD28.

[0190] As used herein, the term “epitope” refers to an antigenic determinant that interacts with a specific antigen-binding site in the variable region of an antibody molecule, known as a paratope. A single antigen may have two or more epitopes. Thus, different antibodies may bind to different regions on an antigen and have different biological effects. Epitopes may be either conformal or linear. Conformal epitopes are generated by spatially juxtaposed amino acids from different segments of a linear polypeptide chain. Linear epitopes are epitopes generated by adjacent amino acid residues within a polypeptide chain. In certain circumstances, epitopes may include sugar, phosphoryl, or sulfonyl groups on an antigen.

[0191] Using various techniques known to those skilled in the art, it is possible to determine whether the antigen-binding domain of an antibody "interacts with one or more amino acids" within a polypeptide or protein. Exemplary techniques include, for example, routine cross-blocking assays as described in Antibodies, Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harb., NY), alanine scanning mutation analysis, peptide blot analysis (Reineke, 2004, Methods Mol Biol 248:443-463), and peptide cleavage analysis. Furthermore, methods such as antigen epitope excision, epitope extraction, and chemical modification can be employed (Tomer, 2000, Protein Science 9:487-496). Another method that can be used to identify the amino acids in the polypeptide with which the antigen-binding domain of an antibody interacts is hydrogen / deuterium exchange detected by mass spectrometry. In general terms, a hydrogen / deuterium exchange method involves labeling the target protein with deuterium, followed by conjugating the antibody to the deuterium-labeled protein. Next, the protein / antibody complex is transferred to water, and hydrogen-deuterium exchange is induced at all residues except those protected by the antibody (which remain deuterium-labeled). After antibody dissociation, the target protein is subjected to protease cleavage and mass spectrometry to identify the deuterium-labeled residues corresponding to the specific amino acids with which the antibody interacts. See, for example, Ehring (1999) Analytical Biochemistry 267(2):252-259 and Engen and Smith (2001) Anal. Chem. 73:256A-265A. X-ray crystallography of the antigen / antibody complex can also be used for epitope mapping purposes.

[0192] This specification provides anti-CD38 antibodies that bind to the same epitopes as any of the specific exemplary antibodies described herein (for example, antibodies comprising any of the amino acid sequences listed in Table 1 of this specification). Similarly, the present invention also includes anti-CD38 antibodies that compete with any of the specific exemplary antibodies described herein (for example, antibodies comprising any of the amino acid sequences listed in Table 1 of this specification) for binding to CD38.

[0193] This specification provides anti-CD28 antibodies that bind to the same epitopes as any of the specific exemplary antibodies described herein (for example, antibodies comprising any of the amino acid sequences listed in Table 4 of this specification). Similarly, the present invention also includes anti-CD28 antibodies that compete with any of the specific exemplary antibodies described herein (for example, antibodies comprising any of the amino acid sequences listed in Table 4 of this specification) for binding to CD28.

[0194] Similarly, provided herein are bispecific antigen-binding molecules comprising a first antigen-binding domain that specifically binds to human CD38 and a second antigen-binding domain that specifically binds to human CD28, wherein the first antigen-binding domain competes with any of the specific exemplary CD38-specific antigen-binding domains described herein for binding to CD38, and / or the second antigen-binding domain competes with any of the specific exemplary CD28-specific antigen-binding domains described herein for binding to CD28.

[0195] Whether a particular antigen-binding molecule (e.g., an antibody) or its antigen-binding domain binds to the same epitope as the reference antigen-binding molecule of the present invention, or competes for binding, can be readily determined using routine methods known in the art. For example, to determine whether a test antibody binds to the same CD38 (or CD28) epitope as the reference bispecific antigen-binding molecule of the present disclosure, the reference bispecific molecule is first bound to the CD38 protein (or CD28 protein). The ability of the test antibody to bind to the CD38 (or CD28) molecule is then evaluated. If the test antibody can bind to CD38 (or CD28) after saturated binding with the reference bispecific antigen-binding molecule, it can be concluded that the test antibody binds to a different CD38 (or CD28) epitope than the reference bispecific antigen-binding molecule. On the other hand, if the test antibody fails to bind to the CD38 (or CD28) molecule after saturated binding with the reference bispecific antigen-binding molecule, the test antibody may bind to the same CD38 (or CD28) epitope as the epitope bound by the reference bispecific antigen-binding molecule. Additional routine experiments (e.g., peptide mutation and binding analysis) can be performed to confirm whether the observed lack of binding of the test antibody is indeed due to binding to the same epitope as the reference bispecific antigen-binding molecule, or whether steric barrier (or another phenomenon) is the cause of the observed lack of binding. This type of experiment can be performed using ELISA, RIA, Biacore, flow cytometry, or any other quantitative or qualitative antibody binding assay available in the art. According to certain embodiments of the present invention, if, as measured by a competitive binding assay, for example, a 1, 5, 10, 20, or 100-fold excess of one antigen-binding protein inhibits the binding of the other by at least 50%, but preferably up to 75%, 90%, or 99%, then the two antigen-binding proteins bind to the same (or overlapping) epitope (see, for example, Junghans et al., Cancer Res. 1990:50:1495-1502).Alternatively, if essentially all amino acid mutations in an antigen that reduce or eliminate the binding of one antigen-binding protein also reduce or eliminate the binding of the other, then the two antigen-binding proteins are considered to bind to the same epitope. If only a subset of amino acid mutations that reduce or eliminate the binding of one antigen-binding protein also reduces or eliminates the binding of the other, then the two antigen-binding proteins are considered to have a "duplicate epitope."

[0196] To determine whether an antibody or its antigen-binding domain competes for binding with a reference anti-antigen-binding molecule, the binding methodology described above is performed in two directions: In the first direction, the reference antigen-binding molecule is bound to the CD38 protein (or CD28 protein) under saturated conditions, and then the binding of the test antibody to the CD38 (or CD28) molecule is evaluated. In the second direction, the test antibody is bound to the CD38 (or CD28) molecule under saturated conditions, and then the binding of the reference antigen-binding molecule to the CD38 (or CD28) molecule is evaluated. In both directions, if only the first (saturated) antigen-binding molecule can bind to the CD38 (or CD28) molecule, it is concluded that the test antibody and the reference antigen-binding molecule compete for binding to the CD38 (or CD28) molecule. As will be understood by those skilled in the art, an antibody that competes for binding with a reference antigen-binding molecule does not necessarily bind to the same epitope as the reference antibody, but can sterically block the binding of the reference antibody by binding to an overlapping or adjacent epitope.

[0197] Preparation of antigen-binding domains and construction of bispecific molecules Antigen-binding domains specific to a particular antigen can be prepared by any antibody production technique known in the art. Once obtained, two different antigen-binding domains specific to two different antigens (e.g., CD38 and CD28) can be appropriately positioned relative to each other to produce the bispecific antigen-binding molecule of the present invention using routine methods. (A consideration of exemplary bispecific antibody formats that can be used to construct the bispecific antigen-binding molecule of the present invention is provided elsewhere in this specification.) In certain embodiments, one or more of the individual components (e.g., heavy and light chains) of the bispecific antigen-binding molecule of the present invention are derived from a chimeric antibody, a humanized antibody, or a fully human antibody. Methods for producing such antibodies are well known in the art. For example, one or more of the heavy and / or light chains of the bispecific antigen-binding molecule of the present invention can be prepared using VELOCIMMUNE® technology. Using VELOCIMMUNE® technology (or any other human antibody production technique), a high-affinity chimeric antibody against a particular antigen (e.g., CD38 or CD28) having a human variable region and a mouse constant region is first isolated. Antibodies are characterized and selected for desirable features, including affinity, selectivity, and epitopes. The mouse constant region is substituted with a desired human constant region to generate a fully human heavy and / or light chain that can be incorporated into the bispecific antigen-binding molecule of the present invention.

[0198] Human bispecific antigen-binding molecules may be produced using genetically modified animals. For example, a genetically modified mouse in which the endogenous mouse immunoglobulin light chain variable sequence cannot be rearranged and expressed may be used, and this mouse expresses only one or two human light chain variable domains encoded by a human immunoglobulin sequence operably linked to the mouse kappa constant gene at the endogenous mouse kappa locus. Using such a genetically modified mouse, a fully human bispecific antigen-binding molecule can be produced containing two different heavy chains that associate with the same light chain, each containing a variable domain derived from one of two different human light chain variable region gene segments. (See, for example, US2011 / 0195454). Fully human means an antibody or its antigen-binding fragment or immunoglobulin domain containing an amino acid sequence encoded by DNA derived from a human sequence throughout the entire length of each polypeptide of the antibody or its antigen-binding fragment or immunoglobulin domain. In some cases, a fully human sequence is derived from a human endogenous protein. In other examples, a fully human protein or protein sequence contains a chimeric sequence in which each component sequence is derived from a human sequence. While not bound by any single theory, chimeric proteins or chimeric sequences are generally designed to minimize the generation of immunogenic epitopes at the junctions of their constituent sequences compared to, for example, any wild-type human immunoglobulin region or domain.

[0199] biological equivalent The present invention encompasses antigen-binding molecules having amino acid sequences that retain the ability to bind to CD38 and / or CD28, although these differ from the exemplary molecules disclosed herein. Such variant molecules may include the addition, deletion, or substitution of one or more amino acids compared to the parent sequence, but exhibit biological activity that is essentially equivalent to the biological activity of the described bispecific antigen-binding molecules.

[0200] The present disclosure includes antigen-binding molecules that are biologically equivalent to any of the exemplary antigen-binding molecules described herein. Two antigen-binding proteins, or antibodies, are considered to be biological equivalents if they are pharmaceutical equivalents or pharmaceutical alternatives that do not show significant differences in the rate and extent of absorption when administered, for example, under similar experimental conditions, at the same molar dose, either as a single dose or multiple doses. Some antigen-binding proteins have the same extent of absorption but different rates of absorption, and such differences in absorption rate are considered intentional and reflected in the labeling, for example, not essential for achieving an effective in vivo drug concentration in chronic use and not medically significant for the specific pharmaceutical being studied, and can be considered biological equivalents and thus equivalents or pharmaceutical alternatives.

[0201] In one embodiment, two antigen-binding proteins are biological equivalents if there are no clinically significant differences in their safety, purity, and potency.

[0202] In one embodiment, two antigen-binding proteins are biological equivalents if a patient can be switched between a reference product and a biological product one or more times without an expected increase in the risk of adverse effects, including a clinically significant change in immunogenicity, compared to continued therapy without switching, or without a decrease in efficacy.

[0203] In one embodiment, two antigen-binding proteins are biological equivalents if they both act by one or more common mechanisms of action for one or more conditions of use, insofar as such mechanisms are known.

[0204] Bioequivalence can be demonstrated by in vivo and in vitro methods. Measurements of bioequivalence include, for example, (a) in vivo studies in humans or other mammals measuring the concentration of an antibody or its metabolite as a function of time in blood, plasma, serum or other biological fluids; (b) in vitro studies that correlate with and reasonably predict human in vivo bioavailability data; (c) in vivo studies in humans or other mammals measuring the appropriate acute pharmacological effect of an antibody (or its target) as a function of time; and (d) well-controlled clinical trials to establish the safety, efficacy, or bioavailability or bioequivalence of an antigen-binding protein.

[0205] Bioequivalent variants of the exemplary bispecific antigen-binding molecules described herein may be constructed, for example, by various substitutions of residues or sequences, or by deletions of terminal or internal residues or sequences that are not required for biological activity. For example, cysteine ​​residues that are not essential for biological activity may be deleted or substituted with other amino acids to prevent the formation of unnecessary or inaccurate intramolecular disulfide crosslinks during regeneration. In other contexts, bioequivalent antigen-binding proteins may include variants of the exemplary bispecific antigen-binding molecules described herein that involve amino acid changes that alter the glycosylation properties of the molecule, such as mutations that eliminate or remove glycosylation.

[0206] Species selectivity and species cross-reactivity According to certain embodiments of the present invention, an antigen-binding molecule is provided that binds to human CD28 but not to CD28 from other species. An antigen-binding molecule is also provided that binds to human CD38 but not to CD38 from other species. The present invention also includes an antigen-binding molecule that binds to human CD28 and CD38 from one or more non-human species, and / or an antigen-binding molecule that binds to human CD28 and CD28 from one or more non-human species.

[0207] According to certain exemplary embodiments of the present invention, antigen-binding molecules are provided that can or cannot bind to one or more of the CD38 and / or CD28 of mice, rats, guinea pigs, hamsters, gerbils, pigs, cats, dogs, rabbits, goats, sheep, cattle, horses, camels, cynomolgus macaques, marmosets, rhesus macaques, or chimpanzees. For example, certain exemplary embodiments disclosed herein provide a bispecific antigen-binding molecule comprising a first antigen-binding domain that binds to human CD38 and cynomolgus macaque CD38 and a second antigen-binding domain that specifically binds to human CD28, or a bispecific antigen-binding molecule comprising a first antigen-binding domain that binds to human CD38 and cynomolgus macaque CD38 and a second antigen-binding domain that specifically binds to human CD28.

[0208] Therapeutic preparations and administration The present invention provides pharmaceutical compositions comprising the antigen-binding molecule of the present invention. The pharmaceutical compositions of the present invention are formulated with suitable carriers, excipients, and other agents that provide improvements in transport, delivery, and tolerability. Numerous suitable formulations can be found in the formulary known to all pharmacists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic)-containing vesicles (e.g., LIPOFECTIN®, Life Technologies, Carlsbad, CA), DNA conjugates, anhydrous absorbent pastes, oil-in-water and water-in-oil emulsions, emulsion carbowaxes (polyethylene glycol of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowaxes. See also Powell et al. "Compendium of excipients for parenteral formulations" PDA (1998) J Pharm Sci Technol 52:238-311.

[0209] The dose of the antigen-binding molecule administered to a patient may vary depending on the patient's age and physique, the target disease, condition, and route of administration. Preferred doses are typically calculated according to body weight or body surface area. When the bispecific antigen-binding molecule of the present invention is used for therapeutic purposes in adult patients, it may be advantageous to administer the bispecific antigen-binding molecule of the present invention intravenously as a single dose at a dose of approximately 0.01 to 20 mg per kg of body weight, more preferably approximately 0.02 to 7 mg, 0.03 to 5 mg, or 0.05 to 3 mg per kg of body weight. The frequency and duration of treatment can be adjusted according to the severity of the condition. Effective doses and schedules for administering the bispecific antigen-binding molecule can be determined empirically, but the patient's progress may be monitored by periodic evaluations, and the dose may be adjusted accordingly. Furthermore, interspecies scaling of doses can be performed using methods well known in the art (e.g., Mordenti et al., 1991, Pharmaceut. Res. 8:1351).

[0210] Various delivery systems are known and can be used to administer the pharmaceutical composition of the present invention. For example, liposomes, microparticles, encapsulation in microcapsules, recombinant cells capable of expressing mutant viruses, receptor-dependent endocytosis, etc. (see, e.g., Wu et al., 1987, J. Biol. Chem. 262:4429-4432). Methods of delivery are not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The composition can be administered by any convenient route, for example, by injection or bolus injection, by absorption via the epithelium or mucocutaneous lining (e.g., oral mucosa, rectal mucosa, and intestinal mucosa), and can be administered together with other bioactive agents. Administration may be systemic or topical.

[0211] The pharmaceutical composition of the present invention can be delivered subcutaneously or intravenously using standard needles and syringes. In addition, with respect to subcutaneous delivery, pen-type delivery devices are readily applicable to the delivery of the pharmaceutical composition of the present invention. Such pen-type delivery devices may be reusable or disposable. Reusable pen-type delivery devices generally utilize replaceable cartridges containing the pharmaceutical composition. Once all of the pharmaceutical composition inside the cartridge has been administered and the cartridge is empty, the empty cartridge can be easily discarded and replaced with a new cartridge containing the pharmaceutical composition. The pen-type delivery device can then be reused. Disposable pen-type delivery devices do not have replaceable cartridges. Rather, disposable pen-type delivery devices are sold pre-filled with the pharmaceutical composition held in a reservoir inside the device. Once the pharmaceutical composition is emptied from the reservoir, the entire device is discarded.

[0212] Numerous reusable pen-type and auto-injector delivery devices have applications for subcutaneous delivery of the pharmaceutical compositions of the present invention. Examples include, but are not limited to, AUTOPEN® (Owen Mumford, Inc., Woodstock, UK), DISETRONIC® pen (Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX 75 / 25® pen, HUMALOG® pen, HUMALIN 70 / 30® pen (Eli Lilly and Co., Indianapolis, IN), NOVOPEN® I, II, and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR® (Novo Nordisk, Copenhagen, Denmark), BD® pen (Becton Dickinson, Franklin Lakes, NJ), OPTIPEN®, OPTIPEN PRO®, and OPTIPEN®. Examples include STARLET (trademark) and OPTICLIK (trademark) (sanofi-aventis, Frankfurt, Germany). Examples of disposable pen-type delivery devices for use in subcutaneous delivery of the pharmaceutical composition of the present invention include, but are not limited to, SOLOSTAR (trademark) pen (sanofi-aventis), FLEXPEN (trademark) (Novo Nordisk), KWIKPEN (trademark) (Eli Lilly), SURECLICK (trademark) Autoinjector (Amgen, Thousand Oaks, CA), PENLET (trademark) (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, LP), and HUMIRA (trademark) Pen (Abbott Labs, Abbott Park, IL).

[0213] In certain circumstances, pharmaceutical compositions may be delivered by controlled-release systems. In one embodiment, a pump may be used (see Langer; Sefton, 1987, CRC Crit.Ref. Biomed.Eng. 14:201 above). In another embodiment, a multimeric material may be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, Florida). In yet another embodiment, the controlled-release system may be positioned near the target of the composition, and therefore only a fraction of the systemic dose may be required (see, for example, Goodson, 1984, Medical Applications of Controlled Release, above, vol.2, pp.115-138). Other controlled-release systems are discussed in the overview by Langer, 1990, Science 249:1527-1533.

[0214] The injectable preparations may include dosage forms for intravenous, subcutaneous, intradermal, and intramuscular injection, intravenous infusion, etc. These injectable preparations may be prepared by known methods. For example, an injectable preparation may be prepared by dissolving, suspending, or emulsifying the antibody or a salt thereof described above in a sterile aqueous or oily medium conventionally used for injection. Examples of aqueous injectable mediums include physiological saline, isotonic solutions containing glucose, and other adjuvants, which may be used in combination with suitable solubilizers such as alcohol (e.g., ethanol), polyalcohols (e.g., propylene glycol, polyethylene glycol), and nonionic surfactants [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)]. Examples of oily mediums include sesame oil and soybean oil, which may be used in combination with solubilizers such as benzyl benzoate and benzyl alcohol. The injectable solutions thus prepared are preferably filled into suitable ampoules.

[0215] Advantageously, the above-described pharmaceutical compositions for oral or parenteral use are prepared into dosage forms in unit doses suitable for adapting the dose of the active ingredient. Such dosage forms in unit doses include, for example, tablets, pills, capsules, injections (ampoules), and suppositories. The amount of the aforementioned antibody contained is generally about 5 to about 500 mg per dosage form in unit doses, and in particular, in the form of injections, the antibody is preferably contained in about 5 to about 100 mg, and in other dosage forms, it is preferably contained in about 10 to about 250 mg.

[0216] Therapeutic use of antigen-binding molecules This disclosure includes a method comprising administering a therapeutic composition comprising an anti-CD38 antibody, an anti-CD28 antibody, or an antigen-binding fragment thereof, or a bispecific antigen-binding molecule that specifically binds to CD38 and CD28, to a subject in need thereof. The therapeutic composition may include any of the antibodies or bispecific antigen-binding molecules disclosed herein, and a pharmaceutically acceptable carrier or diluent. As used herein, the expression “subject in need thereof” means a human or non-human animal exhibiting symptoms or signs of one or more cancers (e.g., a subject exhibiting a tumor or suffering from any of the cancers described below), or otherwise a subject that would benefit from inhibition or reduction of CD38 activity or depletion of CD38+ cells (e.g., multiple myeloma cells).

[0217] The antibodies and bispecific antigen-binding molecules (and therapeutic compositions comprising them) of the present invention are particularly useful for treating any disease or disorder in which stimulation, activation, and / or targeting of an immune response is beneficial. In particular, the anti-CD38 antibody, anti-CD28 antibody, or anti-CD38 × anti-CD28 bispecific antigen-binding molecule of the present invention may be used to treat, prevent, and / or improve any disease or disorder associated with or mediated by the expression or activity of CD38 or / or BCMA, or the proliferation of CD38+ and / or BCMA+ cells. The mechanism by which the therapeutic methods of the present invention are achieved involves killing, for example, cells expressing CD38 in the presence of effector cells, for example, by CDC, apoptosis, ADCC, phagocytosis, or a combination of two or more of these mechanisms. Examples of CD38-expressing cells that can be inhibited or killed using the bispecific antigen-binding molecules of the present invention include, for example, multiple myeloma cells.

[0218] The antibodies and antigen-binding molecules of this disclosure may be used to treat diseases or disorders associated with CD38 expression, including, for example, multiple myeloma, B-cell leukemia, hepatocellular carcinoma, non-small cell lung cancer, melanoma, pancreatic ductal adenocarcinoma, glioma, or breast cancer, or other cancers partially characterized by having CD38+ cells.

[0219] According to certain embodiments, anti-CD38 × anti-CD28 antibodies, or anti-CD38 antibodies, or anti-CD28 antibodies are useful, for example, for treating tumor cells expressing BCMA or CD20. Antigen-binding molecules provided herein may also be used to treat diseases or disorders associated with BCMA expression, including, for example, multiple myeloma or other B-cell or plasma cell carcinomas, such as Waldenström macroglobulinemia, Burkitt lymphoma, and diffuse large B-cell lymphoma, non-Hodgkin lymphoma, chronic lymphocytic leukemia, follicular lymphoma, mantle cell lymphoma, marginal zone lymphoma, lymphoplasmacytic lymphoma, and Hodgkin lymphoma. According to certain embodiments of the present invention, anti-CD38 × anti-CD28 antibodies, or anti-CD38 antibodies, or anti-CD28 antibodies are useful for treating patients with multiple myeloma. According to other related embodiments of the present invention, a method is provided comprising administering an anti-CD38 × anti-CD28 bispecific antibody provided herein in combination with an anti-BCMA antibody, or an anti-BCMA × anti-CD3 bispecific antigen-binding molecule, or an anti-CD20 × anti-CD3 bispecific antigen-binding molecule disclosed herein, to a patient suffering from cancer cells expressing BCMA or CD20. Analytical / diagnostic methods known in the art, such as tumor scanning, may be used to determine whether a patient has multiple myeloma or another B-cell lineage cancer.

[0220] This disclosure also includes methods for treating residual cancer in a subject. As used herein, the term “residual cancer” means the presence or persistence of one or more cancer cells in a subject after treatment with anticancer therapy.

[0221] In certain embodiments, the present invention provides a method for treating a disease or disorder associated with CD38 expression (e.g., multiple myeloma), comprising administering to a subject one or more of the anti-CD38 antibody, anti-CD28 antibody, or bispecific antigen-binding molecules described elsewhere in this specification, after the subject has been determined to have multiple myeloma. For example, this disclosure includes a method for treating multiple myeloma, comprising administering to a patient an anti-CD38 antibody, anti-CD28 antibody, or anti-CD38 × anti-CD28 bispecific antigen-binding molecule one day, two days, three days, five days, six days, or four weeks, two months, four months, six months, eight months, one year, or longer, after the subject has received other immunotherapy or chemotherapy.

[0222] Combination therapies and formulations This disclosure provides a method comprising administering a pharmaceutical composition comprising one of the exemplary antibodies and bispecific antigen-binding molecules described herein in combination with one or more additional therapeutic agents. Examples of exemplary additional therapeutic agents that may be administered in combination with, or in combination with, the antigen-binding molecules of the present invention include, for example, antitumor agents (chemotherapeutic agents including, for example, melphalan, vincristine (Oncovin), cyclophosphamide (Cytoxan), etoposide (VP-16), doxorubicin (Adriamycin), liposomal doxorubicin (Doxil), obendamustine (Treanda), or others known to be effective in treating plasma cell tumors of interest). In some embodiments, the second therapeutic agent comprises a steroid. In some embodiments, the second therapeutic agent comprises a targeted therapy comprising thalidomide, lenalidomide, and bortezomib, which is a therapy approved for treating newly diagnosed patients. Lenalidomide, pomalidomide, bortezomib, carfilzomib, panobinostat, ixazomib, elotuzumab, and daratumumab are examples of second-line treatments effective for relapsed myeloma.

[0223] In some embodiments, the second therapeutic agent is an anti-BCMA×CD3 bispecific antibody. An exemplary anti-BCMA×CD3 bispecific antibody is disclosed in US2020 / 0024356, which is incorporated herein by reference. The exemplary anti-BCMA×CD3 bispecific antibody disclosed in US2020 / 0024356 is REGN5458, which comprises an anti-BCMA binding domain having HCVR / LCVR of SEQ ID NO: 66 / 82 and an anti-CD3 binding domain having HCVR / LCVR of SEQ ID NO: 90 / 82, provided in the US2020 / 0024356 sequence listing. In some embodiments, the second therapeutic agent is an anti-CD20×CD3 bispecific antibody. An exemplary anti-CD20×CD3 bispecific antibody is disclosed in U.S. Patent No. 9,657,102, which is incorporated herein by reference. An exemplary anti-CD20×CD3 bispecific antibody disclosed in U.S. Patent No. 9,657,102 is REGN1979, which comprises an anti-CD20 binding domain having HCVR / LCVR as provided in the sequence listing of U.S. Patent No. 9,657,102, and an anti-CD3 binding domain having HCVR / LCVR as provided in SEQ ID NO. 1242 / 1258.

[0224] In certain embodiments, the second therapeutic agent is a regimen including radiotherapy or stem cell transplantation. In certain embodiments, the second therapeutic agent may be an immunomodulator. In certain embodiments, the second therapeutic agent may be a proteasome inhibitor including bortezomib (Velcade), carfilzomib (Kyprolis), or ixazomib (Ninlaro). In certain embodiments, the second therapeutic agent may be a histone deacetylase inhibitor such as panobinostat (Farydak). In certain embodiments, the second therapeutic agent may be a monoclonal antibody, an antibody-drug conjugate, a bispecific antibody conjugated to an antitumor agent, a checkpoint inhibitor, or a combination thereof. Other agents that may be beneficially administered in combination with the antigen-binding molecule of the present invention include small molecule cytokine inhibitors, as well as cytokine inhibitors comprising antibodies that bind to cytokines such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-9, IL-11, IL-12, IL-13, IL-17, and IL-18, or their respective receptors. The pharmaceutical compositions of the present invention (for example, pharmaceutical compositions comprising the anti-CD38 × anti-CD28 bispecific antigen-binding molecule disclosed herein) may also be administered as part of a therapeutic regimen comprising a combination of one or more therapies selected from: monoclonal antibodies other than those described herein that may interact with different antigens on the surface of plasma cells; bispecific antibodies with one arm binding to an antigen on the surface of tumor cells and the other arm binding to an antigen on T cells; antibody-drug conjugates; bispecific antibodies conjugated with antitumor agents; checkpoint inhibitors, e.g., those targeting PD-1 or CTLA-4; or combinations thereof. In certain embodiments, the checkpoint inhibitor may be selected from PD-1 inhibitors such as pembrolizumab (Keytruda), nivolumab (Opdivo), or semiprimab (REGN2810, having the HCVR / LCVR pair of SEQ ID NO: 162 / 170, see PD-1 inhibitor described in US9,987,500).In certain embodiments, the checkpoint inhibitor may be selected from PD-L1 inhibitors such as atezolizumab (Tecentriq), avelumab (Bavencio), or durvalumab (Imfinzi). In certain embodiments, the checkpoint inhibitor may be selected from CTLA-4 inhibitors such as ipilimumab (Yervoy). Other combinations that may be used in conjunction with the antibody of the present invention are described above.

[0225] The disclosure also includes therapeutic combinations comprising any of the antigen-binding molecules referred to herein and one or more inhibitors of any of the aforementioned cytokines, such as VEGF, Ang2, DLL4, EGFR, ErbB2, ErbB3, ErbB4, EGFRvIII, cMet, IGF1R, B-raf, PDGFR-α, PDGFR-β, FOLH1(PSMA), PRLR, STEAP1, STEAP2, TMPRSS2, MSLN, CA9, uroplakin, or the aforementioned cytokines, wherein the inhibitor is an aptamer, antisense molecule, ribozyme, siRNA, peptide body, nanobody, or antibody fragment (e.g., Fab fragment; F(ab')2 fragment; Fd fragment; Fv fragment; scFv; dAb fragment; or other manipulated molecules such as diabodies, triabodies, tetrabodies, minibodies, and minimal recognition units). The antigen-binding molecule of the present invention may also be administered in combination with and / or co-formulated with antiviral agents, antibiotics, analgesics, corticosteroids, and / or NSAIDs. The antigen-binding molecule of the present invention may also be administered as part of a treatment regimen that includes radiotherapy and / or conventional chemotherapy.

[0226] Additional therapeutic active ingredients may be administered immediately before, simultaneously with, or immediately after the administration of the antigen-binding molecule of the present invention; (for the purposes of this disclosure, such an administration regimen is considered to be an administration of the antigen-binding molecule "combined" with the additional therapeutic active ingredient).

[0227] The present invention includes a pharmaceutical composition in which the antigen-binding molecule of the present invention is co-formulated with one or more additional therapeutic active ingredients described elsewhere in this specification.

[0228] Administration regimen According to certain embodiments of the present invention, multiple doses of an antigen-binding molecule (e.g., an anti-CD38 antibody, an anti-CD28 antibody, or a bispecific antigen-binding molecule that specifically binds to CD38 and CD28) may be administered to a subject over a defined time course. Methods according to this aspect of the present invention include administering multiple doses of the antigen-binding molecule to a subject sequentially. As used herein, “administered sequentially” means that each dose of the antigen-binding molecule is administered to the subject at different times, for example, on different days separated by a predetermined interval (e.g., hours, days, weeks, or months). The present invention includes methods comprising administering a single initial dose of the antigen-binding molecule, then one or more secondary doses of the antigen-binding molecule, and optionally, one or more tertiary doses of the antigen-binding molecule, sequentially to a patient.

[0229] The terms “initial dose,” “secondary dose,” and “tertiary dose” refer to the time series of administration of the antigen-binding molecule of the present invention. Therefore, the “initial dose” is the dose administered at the start of the treatment regimen (also referred to as the “baseline dose”), the “secondary dose” is the dose administered after the initial dose, and the “tertiary dose” is the dose administered after the secondary dose. The initial, secondary, and tertiary doses may all contain the same amount of antigen-binding molecule, but may generally differ in terms of administration frequency. However, in certain embodiments, the amounts of antigen-binding molecule contained in the initial, secondary, and / or tertiary doses may differ from each other during the course of treatment (e.g., adjusted as appropriate). In certain embodiments, two or more doses (e.g., 2, 3, 4, or 5 doses) may be administered as a “loading dose” at the start of the treatment regimen, with subsequent doses administered at a lower frequency (e.g., “maintenance doses”).

[0230] In one exemplary embodiment of this disclosure, each secondary and / or tertiary dose is 1 to 26 of the immediately preceding dose (e.g., 1, 1 1 / 2, 2, 2 1 / 2, 3, 3 1 / 2, 4, 4 1 / 2, 5, 5 1 / 2, 6, 6 1 / 2, 7, 7 1 / 2, 8, 8 1 / 2, 9, 9 1 / 2, 10, 10 1 / 2, 11, 11 1 / 2, 12, 12 1 / 2, 13, 13 1 / 2, 14, 14 1 / 2, 15, 15 1 / 2, 16, 16 1 / 2, 17, 17 1 / 2, 18, 18 1 / 2, 19, 19 1 / 2, 20, 20 1 / 2, 21, 21 1 / 2, 22, 22 1 / 2, 23, 23 1 / 2, 24, 24) It is administered 1 / 2, 25, 25 1 / 2, 26, 26 1 / 2, or more weeks later. When used herein, the phrase "immediately preceding dose" means a sequence of multiple doses, where the dose immediately following that sequence is the dose of the antigen-binding molecule administered to the patient without any doses in between.

[0231] Methods according to this aspect of the Disclosure may include administering to a patient any number of secondary and / or tertiary doses of an antigen-binding molecule (e.g., an anti-CD38 antibody, an anti-CD28 antibody, or a bispecific antigen-binding molecule that specifically binds to CD38 and CD28). For example, in one particular embodiment, the patient is administered only a single secondary dose. In another embodiment, the patient is administered two or more secondary doses (e.g., 2, 3, 4, 5, 6, 7, 8, or more). Similarly, in one particular embodiment, the patient is administered only a single tertiary dose. In another embodiment, the patient is administered two or more tertiary doses (e.g., 2, 3, 4, 5, 6, 7, 8, or more).

[0232] In embodiments including multiple secondary doses, each secondary dose may be administered at the same frequency as the other secondary doses. For example, each secondary dose may be administered to the patient 1 to 2 weeks after the most recent dose. Similarly, in embodiments including multiple tertiary doses, each tertiary dose may be administered at the same frequency as the other tertiary doses. For example, each tertiary dose may be administered to the patient 2 to 4 weeks after the most recent dose. Alternatively, the frequency at which secondary and / or tertiary doses are administered to the patient may change throughout the course of the treatment regimen. The frequency of administration may be adjusted by the physician during the course of treatment according to the individual patient's needs after clinical examinations.

[0233] Diagnostic applications of antibodies The anti-CD38 antibodies of this disclosure may also be used, for example, to detect and / or measure CD38 or CD38-expressing cells in a sample for diagnostic purposes. For example, an anti-CD38 antibody, or a fragment thereof, may be used to diagnose a condition or disease characterized by abnormal expression of CD38 (e.g., overexpression, underexpression, absence of expression, etc.). An exemplary diagnostic assay for CD38 may include, for example, contacting a sample obtained from a patient with the anti-CD38 antibody disclosed herein, wherein the anti-CD38 antibody is labeled with a detectable label or reporter molecule. Alternatively, an unlabeled anti-CD38 antibody may be used for diagnostic purposes in combination with a secondary antibody that is itself detectably labeled. The detectable label or reporter molecule may be, for example, 3 H, 14 C, 32 P, 35 S, or 125 This may be a radioactive isotope such as I, a fluorescent or chemiluminescent moiety such as fluorescein isothiocyanate or rhodamine, or an enzyme such as alkaline phosphatase, β-galactosidase, horseradish peroxidase, or luciferase. Another exemplary diagnostic use of the anti-CD38 antibody described herein is for the non-invasive identification and tracking of tumor cells in a subject. 89 Zr-desferrioxamine labeling, etc. 89This includes Zr-labeled antibodies (e.g., positron emission tomography (PET) imaging). (See, for example, Tavare, R. et al. Cancer Res. 2016 Jan 1;76(1):73-82; and Azad, BB. et al. Oncotarget. 2016 Mar 15;7(11):12344-58.) Specific exemplary assays that can be used to detect or measure CD38 in a sample include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescent cell sequencing (FACS).

[0234] Samples that can be used in the CD38 diagnostic assay according to the present invention include any tissue or body fluid sample obtainable from a patient that contains a detectable amount of CD38 protein or a fragment thereof under normal or pathological conditions. Generally, a baseline or standard CD38 level is first established by measuring the CD38 level in a specific sample obtained from a healthy patient (e.g., a patient without a disease or condition associated with abnormal CD38 levels or activity). This baseline level of CD38 can then be compared to the level of CD38 measured in a sample obtained from an individual suspected of having a CD38-related disease (e.g., a tumor containing CD38-expressing cells) or condition.

[0235] device The present invention also provides a container (e.g., a vial or chromatography column) or injection device (e.g., a syringe, a pre-filled syringe or an auto-injector) containing the bispecific antigen-binding molecule (e.g., its pharmaceutical formulation) described herein. The container or injection device may be packaged in a kit.

[0236] An injection device is a device for introducing a substance into the body of a subject (e.g., a human) via a parenteral route, such as intraocular, intravitreous, intramuscular, subcutaneous, or intravenous. For example, an injection device may be a syringe (e.g., an autoinjector, pre-filled with the pharmaceutical formulation) comprising, for example, a cylinder or barrel for holding the fluid to be injected (e.g., an antibody, or fragments thereof, or a pharmaceutical formulation thereof), a needle for penetrating the skin, blood vessels, or other tissue for injecting the fluid, and a plunger for pushing the liquid out of the cylinder through the needle hole into the body of the subject.

[0237] The pharmaceutical compositions provided herein can be delivered subcutaneously or intravenously using standard needles and syringes. In addition, with respect to subcutaneous delivery, pen-type delivery devices are readily applicable to the delivery of the pharmaceutical compositions of the present invention. Such pen-type delivery devices may be reusable or disposable. Reusable pen-type delivery devices generally utilize replaceable cartridges containing the pharmaceutical composition. Once all of the pharmaceutical composition inside the cartridge has been administered and the cartridge is empty, the empty cartridge can be easily discarded and replaced with a new cartridge containing the pharmaceutical composition. The pen-type delivery device can then be reused. Disposable pen-type delivery devices do not have replaceable cartridges. Rather, disposable pen-type delivery devices are sold pre-filled with the pharmaceutical composition held in a reservoir inside the device. Once the pharmaceutical composition is emptied from the reservoir, the entire device is discarded.

[0238] Numerous reusable pen-type and automated injection device delivery devices are used for subcutaneous delivery of the pharmaceutical composition of the present invention. To give just a few examples, though not limited to them, are AUTOPEN (trademark) (Owen Mumford, Inc., Woodstock, UK), DISETRONIC (trademark) pens (Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX 75 / 25 (trademark) pens, HUMALOG (trademark) pens, HUMALIN 70 / 30 (trademark) pens (Eli Lilly and Co., Indianapolis, India), NOVOPEN (trademark) I, II, and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR (trademark) (Novo Nordisk, Copenhagen, Denmark), BD (trademark) pens (Becton Dickinson, Franklin Lakes, NJ), OPTIPEN (trademark), OPTIPEN PRO (trademark), and OPTIPEN. Examples include STARLET® and OPTICLIK® (sanofi-aventis, Frankfurt, Germany). Examples of disposable pen-type delivery devices that have applications in subcutaneous delivery of the pharmaceutical compositions of this disclosure include, but are not limited to, SOLOSTAR® pen (sanofi-aventis), FLEXPEN® (Novo Nordisk), KWIKPEN® (Eli Lilly), SURECLICK® Autoinjector (Amgen, Thousand Oaks, Calif.), PENLET® (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, LP), and HUMIRA® Pen (Abbott Labs, Abbott Park, Ill.).

[0239] This specification provides a method for administering the bispecific antigen-binding molecules of this disclosure, which includes, for example, introducing the molecules into the body of a subject using an injection device, for example, by injection.

[0240] Method of expression Provided herein are recombinant methods for producing the bispecific antigen-binding molecules or immunoglobulin chains thereof, comprising: (i) introducing one or more polynucleotides encoding an immunoglobulin light chain and / or heavy chain, such as a bispecific antigen-binding molecule, into a host cell, wherein the polynucleotides are in a vector and / or incorporated into the host cell chromosome and / or operably linked to a promoter; (ii) culturing the host cell (e.g., mammal, fungus, Chinese hamster ovary (CHO), Pichia or Pichia pastoris) under conditions favorable for polynucleotide expression; and (iii) optionally isolating the bispecific antigen-binding molecule or immunoglobulin chain from the host cell and / or the culture medium in which the host cell is cultivated. The products of such methods also form part of the present disclosure together with the pharmaceutical compositions thereof.

[0241] In embodiments, methods for producing bispecific antigen-binding molecules include, for example, methods for purifying the molecules by column chromatography, precipitation, and / or filtration. The products of such methods also form part of the disclosure together with the pharmaceutical composition.

[0242] Host cells containing (e.g., in a vector) the bispecific antigen-binding molecules and / or polynucleotides encoding the immunoglobulin chains of such molecules are also part of the present invention. Host cells include, for example, mammalian cells such as Chinese hamster ovary (CHO) cells and fungal cells such as Pichia cells (e.g., P. pastoris). [Examples]

[0243] The following examples are provided to fully disclose to those skilled in the art how the methods and compositions of the present invention are prepared and used, and are not intended to limit the scope of what the inventors consider to be their invention. While efforts have been made to ensure accuracy to the figures used (e.g., quantities, temperatures, etc.), some experimental errors and deviations should be taken into account. Unless otherwise indicated, parts are by weight, molecular weight is average molecular weight, temperature is in degrees Celsius, and pressure is atmospheric pressure or near atmospheric pressure.

[0244] The control antibodies used in Examples 9-12 include CD3-binding negative control bispecificity Ab (H4sH17664D) and CD28-binding negative control bispecificity Ab (bsAb5671).

[0245] Appropriate T cell activation requires two signals: "Signal 1" and "Signal 2". "Signal 1" is induced by binding the T cell receptor (TCR) on T cells to peptide-bonded major histocompatibility complex (MHC) molecules on antigen-presenting cells (APCs). "Signal 2" is T cell activation provided by engaging the costimulatory CD28 receptor on T cells with its ligand, differentiation antigen group 80 or 86 (CD80 / CD86), present on APCs. In other words, "Signal 1" can be provided by treatment with tumor-associated antigen (TAA) × CD3 bispecific antibodies such as CD20 × CD3 bispecific antibody (bsAb1979) or BCMA × CD3 bispecific antibody (bsAb5458).

[0246] The isotype control mentioned throughout is IgG4, which is an isotype control (bsAb1979) for the CD20×CD3 bispecific antibody. P-PVA IsoC-1, also known as semiprimab, and IgG4, an isotype control for semiprimab. P IsoC-2, also known as IsoC-2, is one example.

[0247] Example 1. Production of anti-CD38 antibody and anti-CD28 antibody Anti-human CD38 antibodies were obtained by immunizing genetically modified mice containing DNA encoding the heavy chain and kappa light chain variable regions of human immunoglobulin with CD38-expressing cells or CD38-encoding DNA. The immune response of these antibodies was monitored using CD38-specific immunoassay. When the desired immune response was obtained, splenocytes were collected and fused with mouse myeloma cells to maintain their viability and form hybridoma cell lines. Hybridoma cell lines were screened and selected to identify cell lines that produced CD38-specific antibodies. Using this technique, several anti-CD38 chimeric antibodies (i.e., antibodies possessing both the human variable domain and the mouse constant domain) were obtained. Furthermore, as described in US2007 / 0280945A1, several fully human anti-CD38 antibodies were isolated directly from antigen-positive B cells without fusion with myeloma cells.

[0248] Similarly, anti-CD28 antibodies were obtained by immunizing genetically modified mice containing DNA encoding the human immunoglobulin heavy chain and kappa light chain variable regions with cells expressing CD28 or DNA encoding CD28. The immune response of the antibodies was monitored by CD28-specific immunoassays. When the desired immune response was obtained, splenocytes were harvested and fused with mouse myeloma cells to maintain their viability and form hybridoma cell lines. Hybridoma cell lines were screened and selected to identify cell lines that produced CD28-specific antibodies. Using this technique, several anti-CD28 chimeric antibodies (i.e., antibodies possessing both the human variable domain and the mouse constant domain) were obtained. Furthermore, as described in US2007 / 0280945A1, several fully human anti-CD28 antibodies were isolated directly from antigen-positive B cells without fusion with myeloma cells.

[0249] Antibodies were characterized and selected for desirable features, including affinity and selectivity. Where necessary, the mouse constant region was replaced with a desired human constant region, such as a wild-type or modified IgG1 or IgG4 constant region, to generate fully human anti-CD38 or fully human anti-CD28 antibodies. While the selected constant region may vary depending on the specific application, high affinity antigen-binding and target specificity are characteristics found in the variable region.

[0250] The exemplary anti-CD38 and anti-CD28 antibodies produced according to the method of this embodiment, as well as the specific biological properties of the bispecific antibodies constructed from these antibodies, will be described in detail in the following examples.

[0251] Example 2. Amino acid and nucleic acid sequences of the variable regions of the heavy and light chains of the anti-CD38 antibody. Table 1 shows the variable regions of the heavy and light chains and the CDR amino acid sequence identifiers of the selected anti-CD38 antibodies of the present invention. The corresponding nucleic acid sequence identifiers are listed in Table 2. The complete heavy and light chain amino acid sequences and nucleic acid sequences are provided in Table 3. [Table 1] [Table 2] [Table 3]

[0252] The antibodies provided herein may be of any isotype. For example, the anti-CD38 antibodies of the present invention may include the variable domain and CDR sequence described in Tables 1 and 2, as well as human Fc domains such as isotypes IgG4 and IgG1. For specific applications or experiments, the Fc domain may be a mouse Fc domain. As will be understood by those skilled in the art, an antibody having a particular Fc isotype can be converted to an antibody having a different Fc isotype (for example, an antibody having mouse IgG4 Fc can be converted to an antibody having human IgG1, etc.), but in any case the variable domain (including the CDR)—which is indicated by the numerical identifier shown in Tables 1 and 2—remains the same, and the binding properties are expected to be identical or substantially similar regardless of the nature of the Fc domain.

[0253] Example 3: Amino acid and nucleic acid sequences of the variable regions of the heavy and light chains of the anti-CD28 antibody Table 4 shows the variable regions of the heavy and light chains and the CDR amino acid sequence identifiers of the selected anti-CD28 antibodies of the present invention. The corresponding nucleic acid sequence identifiers are listed in Table 5. The complete heavy and light chain amino acid sequences and nucleic acid sequences are provided in Table 6. [Table 4] [Table 5] [Table 6]

[0254] The antibody of the present invention may be of any isotype. For example, the anti-CD28 antibody of the present invention may include the variable domain and CDR sequence described in Tables 4 and 5, as well as a human Fc domain such as isotype IgG4, IgG1, etc. For specific applications or experiments, the Fc domain may be a mouse Fc domain. As will be understood by those skilled in the art, an antibody having a particular Fc isotype can be converted to an antibody having a different Fc isotype (for example, an antibody having mouse IgG4 Fc can be converted to an antibody having human IgG1, etc.), but in any case the variable domain (including the CDR)—which is indicated by the numerical identifier shown in Tables 4 and 5—remains the same, and the binding properties are expected to be identical or substantially similar regardless of the nature of the Fc domain.

[0255] Example 4: Production of bispecific antibodies that bind to CD38 and CD28 This specification provides bispecific antigen-binding molecules that bind to CD28 and CD38, and such bispecific antigen-binding molecules are also referred to herein as “anti-CD38 × anti-CD28,” “anti-CD28 × anti-CD38,” “anti-CD38 × anti-CD28 bispecific molecule,” “anti-CD38 / anti-CD28,” “CD38 × CD28 bispecific molecule,” or “CD38 × CD28 bsAb.” The anti-CD38 portion of the anti-CD38 × anti-CD28 bispecific molecule is useful for targeting tumor cells expressing CD38, and the anti-CD28 portion of the bispecific molecule is useful for activating T cells. The simultaneous binding of CD38 on tumor cells and CD28 on T cells promotes direct killing (cytolysis) of target tumor cells by activated T cells.

[0256] Bispecific antibodies containing anti-CD38-specific binding domains and anti-CD28-specific binding domains were constructed using a standard methodology in which the heavy and light chains derived from the anti-CD38 antibody were combined with the heavy chain derived from the anti-CD28 antibody. In the exemplified bispecific antibodies, the molecules were constructed using the heavy chain derived from the anti-CD38 antibody, the heavy chain derived from the anti-CD28 antibody, and the common light chain derived from the anti-CD38 antibody. In other examples, bispecific antibodies may be constructed using the heavy chain derived from the anti-CD28 antibody, the heavy chain derived from the anti-CD38 antibody, and an indiscriminate antibody light chain or an antibody light chain known to effectively pair with various heavy chain arms.

[0257] Table 7 outlines the components of the antigen-binding domains of various bispecific antibodies prepared according to this embodiment. [Table 7]

[0258] Example 5: Biacore binding kinetics of anti-CD38 antibody and anti-CD28 antibody Surface plasmon resonance (SPR) kinetics were performed to determine the kinetic parameters of CD38 and CD28 bound to the CD38 × CD28 bispecific antibody.

[0259] For kinetic quantification of CD38, SPR experiments were performed using a Biacore 3000 instrument at 25°C. Antibodies were captured on a CM5 anti-human Fc (bsAb2567) binding surface at a flow rate of 8 uL / min for 37 seconds. Approximately 230 RU of each antibody were captured. Monomeric human CD38 with a c-terminal myc-myc-hexahistiine tag (hCD38.mmH, bsAb3305) at concentrations of 90, 30, 10, 3.33, 1.11, or 0.37 nM was injected onto this surface at a flow rate of 50 uL / min for 5 minutes. Dissociation was measured for 10 minutes. D And t1 / 2 were calculated by fitting the dual-reference sensorgram to a 1:1 coupled model.

[0260] For kinetic quantification of CD28, SPR experiments were performed using a Biacore 3000 instrument at 25°C. Dimeric human CD28 with a c-terminal mouse Fc tag (hCD28.mFc, bsAb2012) was captured on a CM5 anti-mouse Fc-(GE) binding surface at a flow rate of 8 uL / min for 37 seconds. Approximately 150 RU of hCD28.mFc was captured. CD38×CD28 antibodies (bsAb7945 and bsAb6031) at concentrations of 90, 30, 10, 3.33, 1.11, or 0.37 nM were injected onto this surface at a flow rate of 50 uL / min for 5 minutes. Dissociation was measured for 10 minutes. D and t 1 / 2 This was calculated by fitting a dual-reference sensorgram to a 1:1 coupled model.

[0261] Tables 8 and 9 show the binding kinetic parameters of hCD38.mmH and hCD28.mFc binding to two different CD38×CD28 bispecific antibodies of the present invention at 25°C. As shown in Table 8, the two bispecific antibodies bind to human CD38 with a KD of less than approximately 10 nM, and in Table 9, they bind to human CD28 with a KD of less than approximately 26 nM. [Table 8] [Table 9]

[0262] Example 6: T cell activation by CD38 × CD28 bispecific antibody in the presence of CD38+ HEK93 cells or MOLP8 cells endogenously expressing BCMA. As mentioned above, proper T cell activation requires two signals, namely "Signal 1" and "Signal 2". "Signal 1" is induced by the binding of the T cell receptor (TCR) on T cells to peptide-bonded major histocompatibility complex (MHC) molecules on antigen-presenting cells (APCs). "Signal 2" is provided by the engagement of the co-stimulating CD28 receptor on T cells with its ligand, differentiation antigen group 80 or 86 (CD80 / CD86), present on the APC (Martin et al. A 44 kilodalton cell surface homodimer regulates interleukin 2 production by activated human T lymphocytes. Journal of immunology, 1986;136(9):3282-7; June et al. T-cell proliferation involving the CD28 pathway is associated with cyclosporine-resistant interleukin 2 gene expression. Molecular and cellular biology. 1987;7(12):4472-81; Harding et al. CD28-mediated signaling co-stimulates murine T cells and prevents induction of anergy in T-cell clones. Nature. 1992;356(6370):607-9). Therefore, activation of CD28 signaling provides a targeted approach for enhancing existing TCR signaling.

[0263] The CD38 × CD28 bispecific antibodies provided herein are CD38 + Target cells CD28 + Designed to mimic the native ligand of CD28 by cross-linking with T cells, it provides signal 2 to enhance T cell activation in the presence of signal 1, which is provided by an allogeneic response delivered by tumor-associated antigen (TAA) × CD3 bispecific antibody or APC.

[0264] In this example, the ability of a CD38×CD28 bispecific antibody to activate human primary T cells by delivering "signal 2" via CD38 and CD28, as determined by IL2 release, IFNγ release, and T cell proliferation, was evaluated in the presence of human embryonic renal cell carcinoma cell lines (HEK293 / hCD20 / hCD38) engineered to express hCD20 and hCD38, using bsAb1979 (CD20×CD3) as "signal 1". HEK293 cells expressing only hCD20 were included as a control to measure the activity that may occur in the absence of CD38 on the APC. Furthermore, the CD38×CD28 bispecific antibody test included MOLP8, a multiple myeloma cell line that endogenously expresses hCD38. Since MOLP8 cells also endogenously express BCMA, bsAb5458 (BCMA×CD3) was included to function as "signal 1". Notably, unlike HEK293 cells, MOLP8 cells can provide detectable allogeneic stimulation of T cells that function as "signal 1" in the absence of CD3 stimulation provided by bsAb5458.

[0265] Human first generation CD3 + Isolation of T cells: Human peripheral blood mononuclear cells (PBMCs) were isolated and frozen from healthy donor leukocyte packs from Precision for Medicine (Donor 555130) using the EasySep® Direct Human PBMC Isolation Kit, following the manufacturer's recommended protocol. CD3 + T cells are controlled by StemCell Technologies' EasySep® Human CD3 + Using a T Cell Isolation Kit, thawed PBMCs were isolated according to the manufacturer's recommended instructions.

[0266] IL2 and IFNγ release assay: Concentrated CD3 resuspended in stimulating medium + T cells, 1 × 105 The cells were added to a 96-well round-bottom plate at a concentration of cells / well. The growth-arrested HEK293 / hCD20 / hCD38 or HEK293 / hCD20 was then added in 1 × 10⁶ units. 4 CD3 at the final concentration of cells / well + It was added to T cells. MOLP8 cells that had stopped proliferating were added in 5 × 10⁻⁶ units. 4 CD3 at the final concentration of cells / well + The antibodies were added to T cells. After adding the cells, a constant 0.1 nM bsAb1979 or its corresponding isotype control (IsoC-1) was added to wells containing HEK293 / hCD20 / hCD38 or HEK293 / hCD20. A constant 0.5 nM bsAb5458 or IsoC-1 was added to wells containing MOLP8 cells. Subsequently, bsAb6031, bsAb7945, and IsoC-1 were dose-titrated at 3 pM to 200 nM in a 1:4 dilution and added to the wells. The final point of the 10-point dilution did not contain the dose-titrated antibody. The plates were incubated at 37°C in 5% CO2 for 48 hours, and 5 μL of total supernatant was taken and used to measure IL2. After 72 hours, 45 μL of total supernatant was taken, and 5 μL was used to measure IFNγ. The amount of cytokines in the assay supernatant was determined using the PerkinElmer AlphaLisa kit according to the manufacturer's protocol. Cytokine measurements were obtained using the Perkin Elmer Envision multi-label plate reader, and values ​​were reported as pg / mL. All serial dilutions were tested in two ways.

[0267] Antibody EC 50 The values ​​were determined using GraphPad Prism™ software from a four-parameter logistic equation for a 10-point dose-response curve. Maximum IL2 and IFNγ are given as the mean maximum response detected within the tested dose range.

[0268] T-cell proliferation assay: After removing the final supernatant at 72 hours, 0.25 μCi / well of tritium-labeled thymidine was added to the wells, and the plates were incubated for 6 hours. Thymidine, and therefore tritium, is incorporated into the newly synthesized DNA of dividing cells in higher amounts. After 6 hours of incubation, cells were harvested into a 96-well UniFilter plate, and 30 μL of scintillation fluid was added to each well. Tritium uptake was measured as counts per minute (CPM) using a Microplate Scintillation & Luminescence Counter TopCount NXT instrument. All serial dilutions were tested in two ways.

[0269] Antibody EC 50 The values ​​were determined using GraphPad Prism™ software from a four-parameter logistic equation for a 10-point dose-response curve. Maximum CPM is given as the mean maximum response detected within the tested dose range.

[0270] result: HEK293 / hCD20 and HEK293 / hCD20 / hCD38 In the presence of the target and "Signal 1" provided by bsAb1979, CD38×CD28 antibody treatment (bsAb6031 and bsAb7945) resulted in higher cytokine and proliferation responses compared to their corresponding isotype controls, IsoC-1. However, in the absence of either the target or "Signal 1," CD38×CD28 antibody treatment did not enhance cytokine release from T cells or T cell proliferation. See Tables 10, 11, and 12.

[0271] MOLP8 In the presence of allogeneic MOLP8 cells and in the absence of bsAb5458, CD38×CD28 antibody treatment (bsAb6031 and bsAb7945) resulted in a dose-dependent increase in IL2 release and proliferation compared to the corresponding isotype control. "Signal 1" can be provided by allogeneic MOLP8 cells, but the addition of bsAb5458 was also evaluated. Under these conditions, CD38×CD28 antibody treatment (bsAb6031 and bsAb7945) resulted in a dose-dependent increase in IL2 release and IFNg release compared to the corresponding isotype control, IsoC-1. Since proliferation is a sensitivity reading, the addition of bsAb5458 in the absence of CD38×CD28, coupled with the allogeneic stimulation provided by MOLP8 cells, resulted in a saturated signal for proliferation, and the effect of CD38×CD28 was not detected. See Tables 10, 11, and 12.

[0272] Therefore, in the presence of "signal 1," the CD38×CD28 bispecific antibody activates human primary T cells by delivering "signal 2," as determined by a dose-dependent increase in IL2 release, IFNγ release, and T cell proliferation. [Table 10] [Table 11] [Table 12]

[0273] Example 7: Characterization of CD38 × CD28 bispecific antibody combined with semiprimab As mentioned above, the CD38 × CD28 bispecific antibody is CD38 + Target cells CD28 +Designed to mimic the native ligand of CD28 by cross-linking with T cells, it provides a co-stimulatory "signal 2" to enhance T cell activation in the presence of the existing "signal 1". In this case, T cell recognition of the non-self-determining factor on the tumor cell line NALM-6 results in an allogeneic response and provides "signal 1". In addition to the co-stimulatory signal, there is also an inhibitory signal that works to reduce T cell activity. Ligation of programmed cell death protein 1 receptor (PD-1) on T cells with its ligand PD-L1 on APCs leads to the recruitment of phosphatases to the CD28 and TCR complex (Zou and Chen, Inhibitory B7-family molecules in the tumor microenvironment. Nature Reviews Immunology 2008;8:467-477; Francisco et al., The PD-1 pathway in tolerance and autoimmunity. Immunol Rev 2010;236:219-242; Hui et al., T cell costimulatory receptor CD28 is a primary target for PD-1-mediated inhibition. Science. 2017;355(6332):1428-33), thereby counteracting TCR signaling and CD28 stimulation. Blocking PD-1 / PD-L1 interactions with cemiprimab, an antagonist antibody combined with a CD38 x CD28 bispecific antibody, may enhance T cell function and promote the killing of target cells such as cancer cells.

[0274] In this example, the ability of a CD38×CD28 bispecific antibody to activate human primary T cells by engaging CD38 and CD28 and delivering "signal 2," as determined by IL2 and IFNγ release, is demonstrated by CD38 being engineered to express PD-L1. +The evaluation was performed in the presence of human acute lymphoblastic leukemia cancer cell lines (NALM-6 / hPD-L1). NALM-6 cells provide a sufficient allogeneic TCR response to function as "signal 1". The addition of a certain concentration of the PD-1 antagonist antibody, cemiprimab, was also evaluated.

[0275] Human first generation CD3 + Isolation of T cells: Human peripheral blood mononuclear cells (PBMCs) were isolated and frozen from healthy donor leukocyte packs from Precision for Medicine (Donor 555192) using the EasySep® Direct Human PBMC Isolation Kit, following the manufacturer's recommended protocol. CD3 + T cells were isolated by thawing vials of frozen PBMCs. The donor PBMCs were StemCell Technologies' EasySep® Human CD3 + Using the T Cell Isolation Kit, follow the manufacturer's recommended instructions for CD3 + T cells were concentrated.

[0276] IL2 and IFNγ release assay: Concentrated CD3 resuspended in stimulating medium + T cells, 1 × 10 5 Cells / well were added to 96-well round-bottom plates at a concentration of cells / well. NALM-6 cells or NALM-6 cells engineered to express hPD-L1 were added in 5 × 10⁶ wells. 4 CD3 at the final concentration of cells / well +The antibodies were added to T cells. Subsequently, bsAb6031, bsAb7945, and non-TAA×CD28 were dose-set at 0.76 pM to 50 nM in a 1:4 dilution and added to the wells. The final point of the 10-point dilution did not contain the dose-set antibody. After the addition of the dose-set antibody, a constant 20 nM of either semiprimab or its corresponding isotype control (IsoC-2) was added to the wells. The plates were incubated at 37°C and 5% CO2 for 72 hours, and 50 μL of the total supernatant was taken. 5 μL of the collected supernatant was used to measure IL2 and IFNγ. The amount of cytokines in the assay supernatant was determined using the Perkin Elmer AlphaLisa kit according to the manufacturer's protocol. Cytokine measurements were obtained using the Perkin Elmer Envision multi-label plate reader, and values ​​were reported as pg / mL. All serial dilutions were tested in three ways.

[0277] Antibody EC 50 The values ​​were determined using GraphPad Prism™ software from a four-parameter logistic equation for a 10-point dose-response curve. Maximum cytokines are given as the mean maximum response detected within the tested dose range.

[0278] result: In the presence of allogeneic NALM-6 cells or NALM-6 cells engineered to express PD-L1, CD38×CD28 antibody treatment (bsAb6031 and bsAb7945) resulted in a dose-dependent increase in IL-2 release compared to the corresponding isotype control (IsoC-1). Maximum IL-2 release was lower in the NALM-6 / PD-L1 cell condition compared to NALM-6 cells (which do not express PD-L1). The addition of cemiplimab did not affect IL-2 release in the condition using NALM-6 cells that do not express PD-L1. However, in the presence of PD-L1-expressing NALM-6 cells, maximum IL-2 release increased with the addition of cemiplimab compared to the corresponding isotype control (IsoC-2). This suggests that blocking the interaction between PD-1 and PD-L1 may enhance T cell function.

[0279] Therefore, if the homogeneous TCR response provided by CD38+NALM-6 / hPD-L1 cells is sufficiently present to function as "signal 1", the CD38×CD28 bispecific antibody activates human primary T cells by engaging CD38 and CD28 to deliver "signal 2", as determined by the release of IL2 and IFNγ. [Table 13] [Table 14]

[0280] Example 8: CD38-expressing target cells using CD38 × CD28 bispecific antibody on BCMA and human PBMCs The enhancement of BCMA×CD3 target killing by CD38×CD28 was evaluated using a 96-hour cytotoxic assay targeting H929 cells (CD38+ multiple myeloma cell line). In short, human PBMCs were enhanced by 1×10⁶ 6Cells were seeded at a concentration of 30 pM in supplemental RPMI medium and incubated overnight at 37°C to enrich lymphocytes by depleting adherent macrophages, dendritic cells, and some monocytes. The following day, H929 cells were labeled with 1 μM of the fluorescent tracking dye CFDA-SE, and naive PBMCs with depleted adherent cells were labeled with 1 μM of the fluorescent tracking dye CellTrace Violet. Labeled target cells and PBMCs (effector / target cell ratio 4:1) were labeled with serial dilutions (concentration range: 33 nM to 0.71 pM) of CD38 × CD28 bispecific antibody bsAb6031 or bsAb7945, and with 30 pM fixed concentration BCMA × CD3 (bsAb5458) or IgG4 P-PVA Co-incubated with either the isotype control H4sH10154P3 or BCMA×CD3 or IgG4. P-PVA Wells containing [specific cells] were also included. After incubation at 37°C for 96 hours, cells were harvested from the plates and analyzed by FACS on a FACS BD LSRFortessa-X20. After removing the supernatant for cytokine analysis, the cells were washed with cold PBS and stained with LIVE / DEAD Fixable Aqua Dead Cell Stain to identify viable cells. To assess NCI-H929 killing, cells were gated with a live violet-labeled population. The percentage of the viable population was recorded and used to calculate viability. The viability percentage was normalized against the control condition (target cells in the presence of PBMCs only).

[0281] T cell activation was assessed by incubating cells with antibodies directly conjugated against CD2, CD4, CD8, and CD25. The median fluorescence intensity (MFI) of CD25 on CD2+ / CD4+ or CD2+ / CD8+ T cells was reported as a measure of T cell activation. Furthermore, as T cells proliferate, CellTraceViolet is diluted, resulting in a decrease in MFI as measured by FACS. Therefore, T cell proliferation was reported as a decrease in the MFI of CellTraceViolet on CD2+ / CD4+ or CD2+ / CD8+ T cells.

[0282] The supernatant from this assay was collected for cytokine level analysis. The concentrations of IL-17a, IFNγ, TNFα, IL-10, IL-6, IL-4, and IL-2 were analyzed using a Cytometric Bead Array (CBA) kit according to the manufacturer's instructions. Cytokine levels were interpolated from curves generated by the kit's reference materials and reported as pg / mL. Target cell killing, T cell activation, proliferation, and cytokine levels, as well as the EC50 values ​​for maximum cytokine levels, were calculated using four-parameter nonlinear regression analysis in Prism software.

[0283] result: The ability of the co-stimulatory anti-CD38×CD28 bispecific antibodies bsAb6031 and bsAb7954 to enhance H929 target cell killing and T cell activation mediated by the BCMA×CD3 bispecific antibody bsAb5458 was tested. Furthermore, the ability of bsAb6031 and bsAb7954 to mediate target cell and T cell activation in the presence of non-stimulatory isotype controls was evaluated. bsAb6031 and bsAb7954 enhanced 30 pM BCMA×CD3-mediated cytotoxicity in two donors tested at mean EC50s of 5.2 nM and 0.24 nM, respectively, with mean increases in maximum cytotoxicity of 64% and 93%, respectively, compared to cytotoxicity in the presence of 30 pM BCMA×CD3 alone. In the presence of a 30 pM isotype control, bsAb6031 and bsAb7954 mediated modest cytotoxicity with mean increases of 4% and 5%, respectively, compared to the 30 pM isotype control alone (Table 15, Figure 1). The observed target cell lysis was associated with T cell activation, as measured by CD25 upregulation on CD4+ and CD8+ T cells. bsAb6031 and bsAb7954 enhanced 30pM BCMA×CD3-mediated CD4 T cell activation in two donors tested at mean EC50s of 3.1 nM and 0.20 nM, respectively, by a mean increase of 10-fold and 50-fold in maximum CD25 MFI compared to CD25 MFI in the presence of 30pM BCMA×CD3 alone. bsAb6031 and bsAb7954 enhanced 30pM BCMA×CD3-mediated CD8 T cell activation in two donors tested at mean EC50s of 2.3 nM and 0.24 nM, respectively, by a mean increase of 5-fold and 8-fold in maximum CD25 MFI compared to CD25 MFI in the presence of 30pM BCMA×CD3 alone. In the presence of a 30 pM isotype control, bsAb6031 and bsAb7954 mediated CD4+ T cell activation (5-fold and 8-fold, respectively) and CD8+ T cell activation (6.5-fold and 10-fold, respectively) less significantly than the 30 pM isotype control alone. EC50 values ​​could not be obtained (Table 16, Figure 2). Observed target cell lysis was associated with T cell proliferation measured by dilution of CellTrace violet proliferation dye in CD4+ and CD8+ T cells. bsAb6031 enhanced 30 pM BCMA×CD3-mediated CD4 and CD8 T cell proliferation in one of the two donors tested. bsAb7954 enhanced 30 pM BCMA×CD3-mediated CD4 and CD8 T cell proliferation in two donors tested with mean EC50s of 0.59 nM and 0.41 nM, respectively, by mean increases of 62-fold and 69-fold, respectively, compared to proliferation mediated by 30 pM BCMA×CD3 alone. bsAb6031 and bsAb7945 did not induce proliferation of CD4+ or CD8+ T cells in the presence of a 30 pM isotype control (Table 17, Figure 3). Cytokine release mediated by 30 pM BCMA×CD3 was enhanced in the presence of bsAb6031 and bsAb7945. Specifically, bsAb6031 increased the maximum concentrations of IFNg, IL-2, IL-4, IL-10, and TNFa by 2 to 7 times compared to 30 pM BCMA×CD3 alone, but the EC50 could not be calculated. In the presence of a 30 pM isotype control, bsAb6031 doubled the concentrations of IFNg and TNFa compared to the 30 pM isotype control alone. bsAb7954 increased the concentrations of IFNg, IL-2, IL-4, IL-10, and TNFa by 2 to 13 times at the maximum bsAb7954 concentration, and when calculable, the EC50 was 0.21 nM to 1.5 nM (Table 18, Figure 4).

[0284] In summary, co-stimulation increased the efficacy of target cell damage, T cell activation, and cytokine release compared to observations with BCMA × CD3 alone. [Table 15] [Table 16] [Table 17] [Table 18]

[0285] Example 9: BCMA + CD38 + In vivo effect of CD38 x CD28 bispecific antibody combined with 4 mg / kg BCMA x CD3 bispecific antibody against MOLP-8 human multiple myeloma tumor growth. To determine the in vivo antitumor effect of CD38×CD28 bispecific antibody (bsAb) combined with BCMA×CD3bsAb, a xenotumor study was conducted. - Day 13, immunodeficiency NOD.Cg-Prkdc scid Il2rg tm1Wjl / SzJ(NSG) mice (8-10 weeks old, Jackson Labs, catalog number: 005557) were fed 4x10 cells from a normal, healthy donor. 6 Human peripheral blood mononuclear cells (PBMCs) (Reach Bio, catalog number: 0500-301, lot number: 0160506) were injected intraperitoneally. On day 0, mice were subjected to 2 × 10⁶ cells that had been engineered to also express firefly luciferase (MOLP-8-luciferase cells). 6 Individual BCMA + CD38 + MOLP-8 human multiple myeloma tumor cells (DSMZ, catalog number: ACC569) were administered intravenously. Then, mice (n=5 per group) were immediately administered either a CD3-binding negative control bispecificity Ab (H4sH17664D) or BCMA×CD3 (bsAb5458) bsAb at 4 mg / kg, combined with either a CD28-binding negative control bispecificity Ab or CD38×CD28 bsAb (either bsAb6031 or bsAb7945) at 4 mg / kg. These antibodies were administered to the mice a total of three times, two more times on days 7 and 14. Tumor growth was evaluated over 52 days by measuring tumor bioluminescence (BLI) in anesthetized animals. As a positive control, one group of mice (n=5) was given only MOLP-8 rufusherase cells and PBMCs, but no antibodies (PBS-treated group). To measure background BLI levels, one group of mice (n=5) was left untreated and did not receive tumors, PBMCs, or antibodies (tumor-free group).

[0286] Measurement of heterologous tumor growth Tumor volume was measured using BLI imaging. 150 mg / kg of luciferase substrate D-luciferin suspended in PBS was intraperitoneally injected into mice. Five minutes after this injection, BLI imaging was performed on mice under isoflurane anesthesia using the Xenogen IVIS system. Image acquisition was performed with a field of view in D, a subject height of 1.5 cm, a moderate binning level, and an automated exposure time determined by Living Image Software. BLI signals were extracted using Living Image Software. Regions of interest were drawn around each tumor mass, and photon intensity was recorded as total flux (photons / sec - p / sec).

[0287] result: BCMA × CD3 bsAb monotherapy provides a modest antitumor effect, resulting in a reduction in mean BLI levels compared to the control. CD38 × CD28 bsAb 6031 does not induce any activity as monotherapy, while bsAb 7945 exhibits monotherapy activity, slightly reducing mean BLI levels compared to the control. However, any combination of BCMA × CD3 bsAb + CD38 × CD28 bsAb results in lower mean BLI levels than any of the monotherapy combinations. See Tables 19-29 and Figure 5.

[0288] Therefore, these studies demonstrate that monotherapy with either BCMA × CD3bsAb or CD38 × CD28bsAb shows only modest antitumor effects, while combination therapy with BCMA × CD3bsAb + CD38 × CD28bsAb provides a superior, more potent, combined antitumor effect than either therapy alone. [Table 19] [Table 20] [Table 21] [Table 22] [Table 23] [Table 24] [Table 25] [Table 26] [Table 27] [Table 28] [Table 29]

[0289] Example 10: In vivo effect of CD38 x CD28 bispecific antibody combined with BCMA x CD3 bispecific antibody at a dose of 0.4 mg / kg or 0.04 mg / kg on human multiple myeloma tumor growth. To determine the in vivo antitumor effect of CD38 x CD28 bispecific antibody (bsAb) combined with BCMA x CD3 bsAb, xenotumor studies will be conducted using BCMA. + CD38 + This experiment was performed on MOLP-8 human multiple myeloma tumor cells. This experiment was similar to that described in Example 9, except that the dose of BCMA×CD3bsAb was reduced from 4 mg / kg to 0.4 mg / kg or 0.04 mg / kg. Furthermore, the duration of the experiment was shorter in this example (40 days) compared to 52 days in Example 9.

[0290] - Day 12, immunodeficiency NOD.Cg-Prkdc scid Il2rg tm1Wjl / SzJ(NSG) mice (8-10 weeks old, Jackson Labs, catalog number: 005557) were fed 4x10 cells from a normal, healthy donor. 6Human peripheral blood mononuclear cells (PBMCs) (Reach Bio, catalog number: 0500-301, lot number: 0180821) were injected intraperitoneally. On day 0, mice were subjected to 2 × 10⁶ cells that had been engineered to also express firefly luciferase (MOLP-8-luciferase cells). 6 Individual BCMA + CD38 + MOLP-8 human multiple myeloma tumor cells (DSMZ, catalog number: ACC569) were administered intravenously. Mice (n=4-5 per group) were then immediately administered either a CD3-binding negative control bispecific Ab (H4sH17664D, 0.4 mg / kg) or a BCMA×CD3 bsAb (bsAb5458, 0.4 mg / kg or 0.04 mg / kg) at 4 mg / kg in combination with either a CD28-binding negative control bispecific Ab (bsAb5671) or a CD38×CD28 bsAb (bsAb6031 or bsAb7945). These Abs were administered to the mice a total of three times, two more times on days 7 and 14. Tumor growth was evaluated over 40 days by measuring tumor bioluminescence (BLI) in anesthetized animals. As a positive control, one group of mice (n=4) was administered only MOLP-8 rufusherase cells and PBMCs, but no antibodies (PBS-treated group). To measure background BLI levels, one group of mice (n=5) was left untreated and did not receive tumors, PBMCs, or antibodies (tumor-free group).

[0291] Measurement of heterologous tumor growth Tumor volume was measured using BLI imaging. 150 mg / kg of luciferase substrate D-luciferin suspended in PBS was intraperitoneally injected into mice. Five minutes after this injection, BLI imaging was performed on mice under isoflurane anesthesia using the Xenogen IVIS system. Image acquisition was performed with a field of view in D, a subject height of 1.5 cm, a moderate binning level, and an automated exposure time determined by Living Image Software. BLI signals were extracted using Living Image Software. Regions of interest were drawn around each tumor mass, and photon intensity was recorded as total flux (photons / sec - p / sec).

[0292] result: BCMA × CD3 bsAb (either 0.4 mg / kg or 0.04 mg / kg) + CD28-binding negative control bsAb showed a modest but significant antitumor effect, with mean BLI levels decreasing compared to mice treated with CD3-binding negative control bsAb + CD28-binding negative control bsAb (rows 3 and 10 of Table 30, respectively). Treatment with either CD3-binding negative control bsAb + CD38 × CD28 bsAb (bsAb6031 and bsAb7945) moderately and significantly reduced mean BLI levels compared to mice treated with CD3-binding negative control bsAb + CD28-binding negative control bsAb (rows 1 and 2 of Table 30, respectively). However, any combination of BCMA×CD3bsAb (either 0.4 mg / kg or 0.04 mg / kg) + CD38×CD28bsAb (bsAb6031 and bsAb7945) resulted in significantly lower mean BLI measurements than mice treated with BCMA×CD3bsAb + CD28-negative control bsAb (Table 30, rows 8, 9, 15, and 16), mice treated with CD3-negative control bsAb + bsAb6031 (Table 30, rows 6 and 13), or mice treated with CD3-negative control bsAb + bsAb7945 (Table 30, rows 7 and 14). See also Tables 31-39 and Figure 6.

[0293] Therefore, these studies demonstrate that monotherapy with either BCMA × CD3bsAb or CD38 × CD28bsAb shows only modest antitumor effects, while combination therapy with BCMA × CD3bsAb + CD38 × CD28bsAb provides a superior, more potent, combined antitumor effect than either therapy alone. [Table 30-1] [Table 30-2] [Table 31] [Table 32] [Table 33] [Table 34] [Table 35] [Table 36] [Table 37] [Table 38] [Table 39] [Table 40]

[0294] Example 11: BCMA + CD38 +In vivo antitumor effect of CD38 x CD28 bispecific antibody (bsAb) combined with BCMA x CD3 bispecific antibody against WSU-DLCL2 tumor cells To determine the in vivo antitumor effect of CD38×CD28 bispecific antibody (bsAb) combined with BCMA×CD3bsAb, a xenotumor study was conducted. The tumor cell lines used in this experiment were diffuse large B-cell lymphoma cell lines and BCMA. + CD38 + This was the same as Examples 9 and 10 above, except that it was WSU-DLCL2 and only the CD38 / CD28bsAb tested was bsAb6031.

[0295] Day 0: Immunodeficiency NOD.Cg-Prkdc scid Il2rg tm1Wjl / SzJ(NSG) mice (8-10 weeks old, Jackson Labs, catalog number: 005557) were given 3x10 units of 50% Matrigel mixed together. 6 Individual WSU-DLCL2 tumor cells (Diffuse Large B Cell Lymphoma Cell Line, DSMZ, catalog number: ACC575) and 0.5 x 10 cells derived from a normal donor. 6 Each PBMC (Reach Bio, catalog number: 0500-301, lot number: 0180821) was administered by subcutaneous injection. On day 1, mice (n=5 per group) were administered either a CD3-binding negative control bispecific Ab (H4sH17664D) or a BCMA×CD3 bsAb (bsAb5458) at a dose of 4 mg / kg in combination with either a CD28-binding negative control bispecific Ab (bsAb5671) or a CD38×CD28 bsAb6031. These Abs were administered to the mice two more times on days 8 and 14, for a total of three doses. Tumor growth was evaluated up to day 50 by measuring tumor volume.

[0296] Calculation of xenotumor growth and inhibition: To determine the tumor volume using an external caliper, the maximum vertical diameter (length in mm) and maximum horizontal diameter (width in mm) were determined. The tumor volume based on the caliper measurements was calculated using the following formula: Volume (mm)3 ) = (length × width) 2 ) / 2.

[0297] result: BCMA × CD3 bsAb + CD28-negative control bsAb showed a modest antitumor effect with reduced mean tumor size compared to mice treated with CD3-negative control bsAb + CD28-negative control bsAb (two-way ANOVA, p=0.0004 at day 40 and p<0.0001 at days 43, 47, and 50). Treatment with CD38 × CD28 bsAb (bsAb6031) + CD3-negative control bsAb slightly reduced mean tumor size compared to mice treated with CD3-negative control bsAb + CD28-negative control bsAb (two-way ANOVA, p=0.0007 at day 40 and p<0.0001 at days 43, 47, and 50). However, the combination of BCMA×CD3bsAb+CD38×CD28bsAb(bsAb6031) resulted in significantly lower tumor size than mice that received BCMA×CD3bsAb+CD28-negative control bsAb (two-way ANOVA, p=0.0069 at day 40 and p<0.0001 at days 43, 47, and 50), or mice that received CD3-negative control bsAb+CD38×CD28bsAb6031 (two-way ANOVA, p=0.0043 at day 40 and p<0.0001 at days 43, 47, and 50). See Tables 41-54 and Figure 7.

[0298] Therefore, these studies demonstrate that while monotherapy with either BCMA × CD3bsAb or CD38 × CD28bsAb shows a modest antitumor effect, combination therapy with BCMA × CD3bsAb + CD38 × CD28bsAb provides a superior, more potent, combined antitumor effect than either therapy alone. [Table 41] [Table 42] [Table 43] [Table 44] [Table 45] [Table 46] [Table 47] [Table 48] [Table 49] [Table 50] [Table 51] [Table 52] [Table 53] [Table 54]

[0299] Example 12: In vivo antitumor effect of CD38 x CD28 bispecific antibody (bsAb) combined with BCMA x CD3 bsAb against human multiple myeloma tumor cell proliferation using BCMA + CD38 + MOLP-8. To determine the in vivo antitumor effect of CD38×CD28 bispecific antibodies (bsAbs) combined with BCMA×CD3bsAb, a xenotumor study was conducted. Similar to the previous experiments in Examples 9 and 10, this experiment changed the dose of BCMA×CD3bsAb from 4 mg / kg to 0.4 mg / kg, and the doses of bsAb6031 and bsAb7945 were also changed to 0.4 mg / kg. Furthermore, the experiment duration was shorter in this example (44 days) compared to 52 days in Example 9.

[0300] Xenotumor transplantation and measurement - Day 12, immunodeficiency NOD.Cg-Prkdc scid Il2rg tm1Wjl / SzJ(NSG) mice (8-10 weeks old, Jackson Labs, catalog number: 005557) were fed 4x10 cells from a normal, healthy donor. 6 Human peripheral blood mononuclear cells (PBMCs) (Reach Bio, catalog number: 0500-301, lot number: 0180821) were injected intraperitoneally. On day 0, mice were subjected to 2 × 10⁶ cells that had been engineered to also express firefly luciferase (MOLP-8-luciferase cells). 6 Individual BCMA + CD38 +MOLP-8 human multiple myeloma tumor cells (DSMZ, catalog number: ACC569) were administered intravenously. Mice (n=5 per group) were then immediately administered either a CD3-binding negative control bispecific Ab (H4sH17664D) or BCMA×CD3 (bsAb5458) at 0.4 mg / kg, combined with either a CD28-binding negative control bispecific Ab (bsAb5671) at 4 mg / kg, or a CD38×CD28 bsAb (either bsAb6031 or bsAb7945) at 0.4 mg / kg. These Abs were administered to the mice a total of three times, two more times on days 7 and 15. Tumor growth was evaluated over 44 days by measuring tumor bioluminescence (BLI) in anesthetized animals. As a positive control, one group of mice (n=5) was administered only MOLP-8 rufuscherase cells and PBMCs, but no antibodies (PBS-treated group). To measure background BLI levels, one group of mice (n=5) was left untreated and did not receive tumors, PBMCs, or antibodies (tumor-free group).

[0301] Measurement of heterologous tumor growth Tumor volume was measured using BLI imaging. 150 mg / kg of luciferase substrate D-luciferin suspended in PBS was intraperitoneally injected into mice. Five minutes after this injection, BLI imaging was performed on mice under isoflurane anesthesia using the Xenogen IVIS system. Image acquisition was performed with a field of view in D, a subject height of 1.5 cm, a moderate binning level, and an automated exposure time determined by Living Image Software. BLI signals were extracted using Living Image Software. Regions of interest were drawn around each tumor mass, and photon intensity was recorded as total flux (photons / sec - p / sec).

[0302] result: BCMA × CD3 bsAb + CD28-negative control bsAb showed a modest antitumor effect, with a decrease in mean BLI measurements compared to mice treated with CD3-negative control bsAb + CD28-negative control bsAb. Treatment with either CD3-negative control bsAb + CD38 × CD28 bsAb (bsAb6031 and bsAb7945) slightly decreased mean BLI measurements compared to mice treated with CD3-negative control bsAb + CD28-negative control bsAb. However, any combination of BCMA × CD3 bsAb + CD38 × CD28 bsAb (bsAb6031 and bsAb7945) resulted in lower mean BLI measurements than mice treated with BCMA × CD3 bsAb + CD28-negative control bsAb, mice treated with CD3-negative control bsAb + bsAb6031, or mice treated with CD3-negative control bsAb + bsAb7945. Please refer to Tables 55-64 and Figure 8.

[0303] Therefore, these studies demonstrate that monotherapy with either BCMA×CD3bsAb or CD38×CD28bsAb shows only modest antitumor effects, while combination therapy with BCMA×CD3bsAb + CD38×CD28bsAb provides a more powerful, combined antitumor effect than either therapy alone. [Table 55] [Table 56] [Table 57] [Table 58] [Table 59] [Table 60] [Table 61] [Table 62] [Table 63] [Table 64]

[0304] Example 13: CD38 Cross-Competitive Analysis A competitive binding assay was performed to evaluate the ability of the CD38×CD28 bispecific antibody, CD38 parent antibody, and CD38 comparative antibody to compete with each other for binding to hCD38.mmh.

[0305] The entire experiment was performed at 25°C at a flow rate of 1000 rpm in Octet HBS-EP buffer (pH 7.4 + 1 mg / mL BSA). To evaluate whether the two antibodies were able to compete with each other for binding to their respective epitopes on recombinant human CD38 expressed with a C-terminal myc-myc-hexahistidine tag (hCD38-mmH), approximately 0.27 nm of hCD38-mmH was captured by immersing a high-density anti-His1K coated Octet tip in a well containing 50 ug / mL (300 nM) of hCD38-mmH for 5 minutes. The antigen-coated sensor tip was then placed in a well containing a 50 μg / mL solution of the first anti-CD38 monoclonal antibody or bispecific antibody for 5 minutes to saturate the hCD38-mmH surface. The sensor tip was then immersed in a well containing a 50 μg / mL solution of the second anti-CD38 monoclonal antibody or bispecific antibody. The sensor chip was washed with Octet HBS-EP buffer throughout all steps of the experiment. Real-time binding responses were monitored during the experiment, and binding responses were recorded at the end of each step. The antibody binding responses to pre-complexed hCD38.mmH with the first antibody were compared to determine the competitive / non-competitive behavior of different anti-CD38 monoclonal and bispecific antibodies.

[0306] Results: Data not shown indicate that CD38×CD28bsAb7945 and bsAb6031 compete bidirectionally with each other for binding to hCD38.mmh. The parental anti-CD38 antibody also showed bidirectional cross-competition for binding to hCD38.mmh.

[0307] Example 14: BCMA + CD38 + In vivo antitumor effect of CD38 x CD28 bispecific antibody (bsAb) combined with BCMA x CD3 bsAb against WSU-DLCL2 tumor cell proliferation. To determine the in vivo antitumor effect of CD38×CD28 bispecific antibodies (bsAbs) combined with BCMA×CD3bsAb, the following xenotumor studies were conducted. This example is similar to the previous experiment in Example 11, but this experiment also investigated bsAb6031 and bsAb7945 at doses of 0.4 mg / kg and 0.04 mg / kg.

[0308] Transplantation and measurement of xenotumors. Day 0: Immunodeficiency NOD.Cg-Prkdc scid Il2rg tm1Wjl / SzJ(NSG) mice were given 3x10 units mixed together in 50% Matrigel. 6 Individual BCMA + CD38 + WSU-DLCL2 tumor cells and 0.5x10 6 1 normal donor-derived PBMCs were subcutaneously injected. On day 1, mice (n=5 per group) were administered either a CD3-binding negative control bispecific Ab (H4sH17664D) or a BCMA×CD3 bsAb (bsAb5458) at 0.4 mg / kg, in combination with a CD28-binding negative control bispecific Ab (bsAb5671) at 4 mg / kg, or a CD38×CD28 bsAb (either bsAb6031 or bsAb7945) at 4 mg / kg, 0.4 mg / kg, or 0.04 mg / kg. These Abs were administered to the mice two more times on days 8 and 15, for a total of three doses. Tumor growth was evaluated up to day 50 by measuring tumor volume.

[0309] Calculation of xenotumor growth and inhibition: To determine the tumor volume using an external caliper, the maximum vertical diameter (length in mm) and maximum horizontal diameter (width in mm) were determined. The tumor volume based on the caliper measurements was calculated using the following formula: Volume (mm) 3 ) = (length × width) 2 ) / 2.

[0310] result: BCMA × CD3 bsAb + CD28-binding negative control bsAb showed a modest antitumor effect with reduced mean tumor size compared to mice treated with CD3-binding negative control bsAb + CD28-binding negative control bsAb (p<0.0001 at day 50 by two-way ANOVA). Treatment with CD38 × CD28 bsAb 6031 + CD3-binding negative control bsAb slightly reduced mean tumor size compared to mice treated with CD3-binding negative control bsAb + CD28-binding negative control bsAb (p<0.0089 at day 50 by two-way ANOVA). Treatment with CD38 × CD28 bsAb 7945 + CD3-binding negative control bsAb also slightly reduced mean tumor size compared to mice treated with CD3-binding negative control bsAb + CD28-binding negative control bsAb (p<0.0001 at day 50 by two-way ANOVA). However, the combination of BCMA × CD3 bsAb + 4 mg / kg with CD38 × CD28 bsAb 6031 resulted in significantly lower tumor size than mice treated with BCMA × CD3 bsAb + CD28-negative control bsAb (p<0.0001 at day 50 by two-way ANOVA) or mice treated with CD3-negative control bsAb + CD38 × CD28 bsAb 6031 (p<0.0001 at day 50 by two-way ANOVA). Furthermore, the combination of BCMA×CD3bsAb + CD38×CD28bsAb7945 at 4 mg / kg and 0.4 mg / kg resulted in significantly lower tumor size than mice treated with BCMA×CD3bsAb + CD28-negative control bsAb (p<0.0001 at day 50 by two-way ANOVA for both doses) or mice treated with CD3-negative control bsAb + CD38×CD28bsAb6031 (p<0.0001 at day 50 by two-way ANOVA for both doses). See Figure 9 and Tables 65-78.

[0311] These studies have confirmed that monotherapy with either BCMA × CD3bsAb or CD38 × CD28bsAb shows a modest antitumor effect, while combination therapy with BCMA × CD3bsAb + CD38 × CD28bsAb provides a more powerful, combined antitumor effect than either therapy alone. [Table 65] [Table 66] [Table 67] [Table 68] [Table 69] [Table 70] [Table 71] [Table 72] [Table 73] [Table 74] [Table 75] [Table 76] [Table 77] [Table 78]

[0312] Example 15: In vivo antitumor effect of CD38 x CD28 bispecific antibody (bsAb) combined with BCMA x CD3 bsAb against human multiple myeloma tumor cell proliferation using BCMA + CD38 + MOLP-8. To determine the in vivo antitumor effect of a CD38×CD28 bispecific antibody (bsAb) combined with BCMA×CD3bsAb, a xenotumor study was conducted. This example was similar to the experiment in Example 12, but this experiment tested more mice (10-13 mice per group compared to 5 mice per group in Example 12) and the study period was longer (48 days compared to 44 days in Example 12).

[0313] Xenotumor transplantation and measurement -On day 12, the immunodeficient NOD.Cg-Prkdc scid Il2rg tm1Wjl / SzJ(NSG) mice were given 4x10⁶ mice derived from normal, healthy donors. 6 Human peripheral blood mononuclear cells (PBMCs) were injected intraperitoneally. On day 0, 2 × 10⁶ mice were modified to also express firefly luciferase (MOLP-8-luciferase cells). 6 Individual BCMA + CD38 + MOLP-8 human multiple myeloma tumor cells were administered intravenously. Then, mice (n=10-13 per group) were immediately administered either a CD3-binding negative control bispecific Ab (H4sH17664D) or BCMA×CD3 (bsAb5458) bsAb at 0.4 mg / kg, combined with either a CD28-binding negative control bispecific Ab (bsAb5671) or CD38×CD28 bsAb (either bsAb6031 or bsAb7945) at 0.4 mg / kg. These Abs were administered to the mice two more times on days 7 and 14, for a total of three doses. Tumor growth was evaluated over 48 days by measuring tumor bioluminescence (BLI) in anesthetized animals. As a positive control, one group of mice (n=5) was given only MOLP-8 rufusherase cells and PBMCs, but no antibody (PBS-treated group). To measure background BLI levels, one group of mice (n=5) was left untreated and did not receive tumors, PBMCs, or antibodies (tumor-free group).

[0314] Measurement of heterologous tumor growth Tumor volume was measured using BLI imaging. 150 mg / kg of luciferase substrate D-luciferin suspended in PBS was intraperitoneally injected into mice. Five minutes after this injection, BLI imaging was performed on mice under isoflurane anesthesia using the Xenogen IVIS system. Image acquisition was performed with a field of view in D, a subject height of 1.5 cm, a moderate binning level, and an automated exposure time determined by Living Image Software. BLI signals were extracted using Living Image Software. Regions of interest were drawn around each tumor mass, and photon intensity was recorded as total flux (photons / sec - p / sec).

[0315] result: BCMA × CD3 bsAb + CD28-binding negative control bsAb showed some antitumor effect, with a decrease in mean BLI measurements, compared to mice treated with CD3-binding negative control bsAb + CD28-binding negative control bsAb (two-way ANOVA, p<0.0001 on days 20, 23, and 27). Treatment with CD3-binding negative control bsAb + CD38 × CD28 bsAb (bsAb7945) slightly reduced mean BLI measurements compared to mice treated with CD3-binding negative control bsAb + CD28-binding negative control bsAb (two-way ANOVA, p<0.0001 on day 23). However, all three dose combinations of BCMA×CD3bsAb+CD38×CD28bsAb (bsAb7945) resulted in lower mean BLI measurements than mice administered with BCMA×CD3bsAb+CD28-binding negative control bsAb (two-way ANOVA on day 44: p<0.0001 for 4 mg / kg dose bsAb7945, 0=0.0007 for 0.4 mg / kg dose bsAb7945, and p=0.0018 for 0.04 mg / kg dose bsAb7945; p<0.0001 for all three doses of bsAb7945 on day 48 by two-way ANOVA). See Figure 10 and Tables 79-90.

[0316] Therefore, these studies demonstrate that monotherapy with either BCMA×CD3bsAb or CD38×CD28bsAb shows only modest antitumor effects, while combination therapy with BCMA×CD3bsAb + CD38×CD28bsAb provides a more powerful, combined antitumor effect than either therapy alone. [Table 79] [Table 80] [Table 81] [Table 82] [Table 83] [Table 84] [Table 85] [Table 86] [Table 87] [Table 88] [Table 89] [Table 90]

[0317] The present invention is not limited to the scope of the specific embodiments described herein. In fact, various modifications of the present invention will be apparent to those skilled in the art from the above description and accompanying drawings, in addition to those described herein. Such modifications are intended to fall within the scope of the appended claims.

Claims

1. (a) A first antigen-binding domain comprising a heavy chain variable region (HCVR) and a light chain variable region (LCVR) that specifically binds to human CD38, The HCVR includes three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) within the HCVR, which include the amino acid sequence of SEQ ID NO: 32, and The LCVR comprises a first antigen-binding domain, which includes three light chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3) within the LCVR, which contain the amino acid sequence of SEQ ID NO:

48. (b) An isolated bispecific antigen-binding molecule comprising a second antigen-binding domain that specifically binds to human CD28.

2. The isolated bispecific antigen-binding molecule according to claim 1, wherein the HCVR of the first antigen-binding domain comprises HCDR1 containing the amino acid sequence of SEQ ID NO: 34, HCDR2 containing the amino acid sequence of SEQ ID NO: 36, and HCDR3 containing the amino acid sequence of SEQ ID NO:

38.

3. The isolated bispecific antigen-binding molecule according to claim 1 or 2, wherein the LCVR of the first antigen-binding domain comprises LCDR1 containing the amino acid sequence of SEQ ID NO: 50, LCDR2 containing the amino acid sequence of SEQ ID NO: 52, and LCDR3 containing the amino acid sequence of SEQ ID NO:

54.

4. The isolated bispecific antigen-binding molecule according to claim 1, wherein the first antigen-binding domain comprises an HCVR containing the amino acid sequence of SEQ ID NO: 32 and an LCVR containing the amino acid sequence of SEQ ID NO:

48.

5. The second antigen-binding domain comprises HCVR and LCVR, The HCVR includes three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) within the HCVR, which contain the amino acid sequence of SEQ ID NO:

40. The LCVR includes three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) within the LCVR, which contain the amino acid sequence of SEQ ID NO:

48. An isolated bispecific antigen-binding molecule according to any one of claims 1 to 4.

6. The isolated bispecific antigen-binding molecule according to claim 5, wherein the HCVR of the second antigen-binding domain comprises HCDR1 containing the amino acid sequence of SEQ ID NO: 42, HCDR2 containing the amino acid sequence of SEQ ID NO: 44, and HCDR3 containing the amino acid sequence of SEQ ID NO:

46.

7. The isolated bispecific antigen-binding molecule according to claim 5 or 6, wherein the LCVR of the second antigen-binding domain comprises LCDR1 containing the amino acid sequence of SEQ ID NO: 50, LCDR2 containing the amino acid sequence of SEQ ID NO: 52, and LCDR3 containing the amino acid sequence of SEQ ID NO:

54.

8. The isolated bispecific antigen-binding molecule according to claim 5, wherein the second antigen-binding domain comprises an HCVR containing the amino acid sequence of SEQ ID NO: 40 and an LCVR containing the amino acid sequence of SEQ ID NO:

48.

9. The isolated bispecific antigen-binding molecule according to any one of claims 1 to 8, wherein the first antigen-binding domain binds to CD38 with a dissociation constant (KD) of less than 10 nM as measured by a surface plasmon resonance assay.

10. The isolated bispecific antigen-binding molecule according to any one of claims 1 to 9, wherein the second antigen-binding domain binds to CD28 with a dissociation constant (KD) of less than 26 nM as measured by a surface plasmon resonance assay.

11. The bispecific antigen-binding molecule has the following characteristics: (a) Activating human primary T cells as determined by increased IL2 release, IFNg release, and T cell proliferation, (b) Enhancement of target cell cytotoxicity mediated by BCMA × CD3 bispecific antibodies, (c) To enhance target cell lysis associated with T cell activation, as measured by CD25 upregulation on CD4+ and CD8+ T cells, mediated by a BCMA x CD3 bispecific antibody. (d) To enhance target cell lysis associated with T cell proliferation, as measured by dye dilution in CD4+ and CD8+ T cells, mediated by a BCMA x CD3 bispecific antibody. (e) Enhancement of cytokine release mediated by BCMA x CD3 bispecific antibodies, (f) In a mouse model of multiple myeloma, exhibiting a reduction in tumor burden mediated by a BCMA x CD3 bispecific antibody; or (g) In a mouse multiple myeloma model, exhibiting one or more of the following: an isolated bispecific antigen-binding molecule according to any one of claims 1 to 10.

12. The isolated bispecific antigen-binding molecule according to any one of claims 1 to 11, wherein the isolated bispecific antigen-binding molecule increases IL2 release in CD38+ cells at an EC50 of less than 1 nM.

13. The isolated bispecific antigen-binding molecule according to any one of claims 1 to 11, wherein the isolated bispecific antigen-binding molecule increases IFNg release in CD38+ cells at an EC50 of less than 750 pM.

14. The isolated bispecific antigen-binding molecule according to any one of claims 1 to 11, wherein the isolated bispecific antigen-binding molecule increases the proliferation of CD38+ cells at an EC50 of less than 150 pM.

15. The isolated bispecific antigen-binding molecule according to any one of claims 1 to 14, wherein the isolated bispecific antigen-binding molecule inhibits the proliferation of tumor cells expressing CD38 at a dose of 0.04 mg / kg to 4.0 mg / kg.

16. The isolated bispecific antigen-binding molecule according to any one of claims 1 to 15, wherein the isolated bispecific antigen-binding molecule inhibits the proliferation of CD38+ tumor cells selected from the group consisting of myeloma cells, leukemia cells, hepatocellular carcinoma cells, non-small cell lung cancer cells, melanoma cells, pancreatic ductal adenocarcinoma cells, glioma cells, and breast cancer cells.

17. The isolated bispecific antigen-binding molecule according to any one of claims 1 to 15, wherein the isolated bispecific antigen-binding molecule inhibits the proliferation of CD38+ tumor cells selected from the group consisting of H929 cells, MOLP-8 cells, and WSU-DLCL2 tumor cells.

18. (a) A first antigen-binding domain that specifically binds to human CD38, comprising HCVR and LCVR, wherein the HCVR comprises the HCDR1 amino acid sequence of SEQ ID NO: 34, the HCDR2 amino acid sequence of SEQ ID NO: 36, and the HCDR3 amino acid sequence of SEQ ID NO: 38, (b) A second antigen-binding domain that specifically binds to human CD28, comprising HCVR and LCVR, wherein the HCVR comprises the HCDR1 amino acid sequence of SEQ ID NO: 42, the HCDR2 amino acid sequence of SEQ ID NO: 44, and the HCDR3 amino acid sequence of SEQ ID NO: 46, (c) An isolated bispecific antigen-binding molecule comprising an LCVR containing the LCDR1 amino acid sequence of SEQ ID NO: 50, the LCDR2 amino acid sequence of SEQ ID NO: 52, and the LCDR3 amino acid sequence of SEQ ID NO:

54.

19. (a) A first antigen-binding domain comprising an HCVR containing the amino acid sequence of SEQ ID NO: 32, (b) The isolated bispecific antigen-binding molecule according to claim 18, comprising a second antigen-binding domain comprising an HCVR having the amino acid sequence of SEQ ID NO:

40.

20. The isolated bispecific antigen-binding molecule according to claim 19, further comprising the LCVR amino acid sequence of Sequence ID No.

48.

21. The isolated bispecific antigen-binding molecule according to any one of claims 1 to 20, wherein the molecule is a bispecific antibody.

22. The isolated bispecific antigen-binding molecule according to claim 21, wherein the bispecific antibody is a human antibody.

23. The isolated bispecific antigen-binding molecule according to claim 21, wherein the bispecific antibody is an immunoglobulin molecule comprising four polypeptide chains interconnected by disulfide bonds, i.e., two heavy chains and two light chains, each heavy chain comprising a heavy chain variable region and a heavy chain constant region, and each light chain comprising a light chain variable region and a light chain constant region.

24. The isolated bispecific antigen-binding molecule according to claim 21, wherein the bispecific antibody comprises a first heavy chain and a second heavy chain, and the two heavy chains include a human IgG heavy chain constant region.

25. The isolated bispecific antigen-binding molecule according to claim 24, wherein the constant region of the human IgG heavy chain is isotype IgG1.

26. The isolated bispecific antigen-binding molecule according to claim 24, wherein the constant region of the human IgG heavy chain is isotype IgG4.

27. ​​The bispecific antigen-binding molecule according to claim 21, wherein the bispecific antibody comprises a first heavy chain and a second heavy chain, and only one of the first or second heavy chain contains a C H 3 domain including the H435R (according to EU numbering rules) modification and the Y436F (according to EU numbering rules) modification.

28. The aforementioned bispecific antibody, compared to the wild-type hinge of the same isotype, [Math 1] An isolated bispecific antigen-binding molecule according to any one of claims 21 to 27, comprising a chimeric hinge that reduces receptor binding.

29. The isolated bispecific antigen-binding molecule according to any one of claims 1 to 28, wherein the first antigen-binding domain comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 56 and a light chain containing the amino acid sequence of SEQ ID NO:

60.

30. The isolated bispecific antigen-binding molecule according to any one of claims 1 to 29, wherein the second antigen-binding domain comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 58 and a light chain containing the amino acid sequence of SEQ ID NO:

60.

31. An isolated recombinant antibody that specifically binds to human CD38, wherein the antibody is (a) Heavy chain variable region (HCVR) and light chain variable region (LCVR), wherein the HCVR includes three heavy chain complementarity determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) in the HCVR amino acid sequence of SEQ ID NO: 32, and the LCVR includes three light chain CDRs (LCDR1, LCDR2, and LCDR3) in the LCVR amino acid sequence of SEQ ID NO: 48, HCVR and LCVR, (b) HCVR including the HCDR1 amino acid sequence of SEQ ID NO: 34, the HCDR2 amino acid sequence of SEQ ID NO: 36, and the HCDR3 amino acid sequence of SEQ ID NO: 38, and LCVR including the LCDR1 amino acid sequence of SEQ ID NO: 50, the LCDR2 amino acid sequence of SEQ ID NO: 52, and the LCDR3 amino acid sequence of SEQ ID NO: 54, and (c) HCVR amino acid sequence including SEQ ID NO: 32, and LCVR amino acid sequence including SEQ ID NO: 48, An isolated recombinant antibody containing a sequence selected from the group consisting of the following.

32. A pharmaceutical composition comprising an isolated bispecific antigen-binding molecule according to any one of claims 1 to 30, or an antibody according to claim 31, and a pharmaceutically acceptable carrier or diluent.

33. A nucleic acid molecule comprising a polynucleotide sequence encoding the heavy chain variable region (HCVR) and a polynucleotide sequence encoding the light chain variable region (LCVR) of an isolated antibody that binds to human CD38, wherein the antibody is The three heavy chain complementarity-determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) contained within the HCVR sequence described in Sequence ID No. 32; and The three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within the LCVR sequence described in Sequence ID No. 48 Nucleic acid molecules, including those mentioned above.

34. An expression vector comprising the nucleic acid molecule described in claim 33.

35. A host cell comprising the expression vector described in claim 34.

36. A method for producing the antibody according to claim 31, comprising culturing the host cell according to claim 35 under conditions that enable the production of the antibody.

37. A set of expression vectors comprising a first expression vector and a second expression vector, The first expression vector comprises a polynucleotide sequence encoding the HCVR of an anti-CD38 antibody, comprising HCDR1 containing the amino acid sequence of SEQ ID NO: 34, HCDR2 containing the amino acid sequence of SEQ ID NO: 36, and HCDR3 containing the amino acid sequence of SEQ ID NO: 38; The second expression vector is a set of expression vectors comprising a polynucleotide sequence encoding the LCVR of an anti-CD38 antibody, including LCDR1 containing the amino acid sequence of SEQ ID NO: 50, LCDR2 containing the amino acid sequence of SEQ ID NO: 52, and LCDR3 containing the amino acid sequence of SEQ ID NO:

54.

38. A host cell comprising the set of expression vectors described in claim 37.

39. A method for producing the antibody according to claim 31, comprising culturing the host cell according to claim 38 under conditions that enable the production of the antibody.

40. A set of expression vectors comprising a first expression vector, a second expression vector, and a third expression vector, The first expression vector comprises a polynucleotide sequence encoding a first HCVR of a bispecific antigen-binding molecule, comprising HCDR1 containing the amino acid sequence of SEQ ID NO: 34, HCDR2 containing the amino acid sequence of SEQ ID NO: 36, and HCDR3 containing the amino acid sequence of SEQ ID NO: 38; The second expression vector comprises a polynucleotide sequence encoding a second HCVR of a bispecific antigen-binding molecule, comprising HCDR1 containing the amino acid sequence of SEQ ID NO: 42, HCDR2 containing the amino acid sequence of SEQ ID NO: 44, and HCDR3 containing the amino acid sequence of SEQ ID NO: 46; A set of expression vectors comprising a third expression vector containing a polynucleotide sequence encoding the bispecific antigen-binding molecule LCVR, which includes LCDR1 containing the amino acid sequence of SEQ ID NO: 50, LCDR2 containing the amino acid sequence of SEQ ID NO: 52, and LCDR3 containing the amino acid sequence of SEQ ID NO:

54.

41. A host cell comprising the set of expression vectors described in claim 40.

42. A method for producing a bispecific antigen-binding molecule according to any one of claims 1 to 30, comprising culturing a host cell according to claim 41 under conditions that enable the production of the bispecific antigen-binding molecule.

43. A composition comprising a first nucleic acid molecule and a second nucleic acid molecule, The first nucleic acid molecule comprises a polynucleotide sequence encoding an HCVR of an isolated antibody that binds to human CD38, wherein the HCVR comprises HCDR1 containing the amino acid sequence of SEQ ID NO: 34, HCDR2 containing the amino acid sequence of SEQ ID NO: 36, and HCDR3 containing the amino acid sequence of SEQ ID NO: 38; The second nucleic acid molecule contains a polynucleotide sequence encoding the LCVR of an antibody that binds to human CD38, wherein the LCVR comprises LCDR1 containing the amino acid sequence of SEQ ID NO: 50, LCDR2 containing the amino acid sequence of SEQ ID NO: 52, and LCDR containing the amino acid sequence of SEQ ID NO:

54. A composition containing 3.

44. A composition comprising a first nucleic acid molecule, a second nucleic acid molecule, and a third nucleic acid molecule, The first nucleic acid molecule comprises a polynucleotide sequence encoding a first HCVR of a bispecific antigen-binding molecule, comprising HCDR1 containing the amino acid sequence of SEQ ID NO: 34, HCDR2 containing the amino acid sequence of SEQ ID NO: 36, and HCDR3 containing the amino acid sequence of SEQ ID NO: 38; The second nucleic acid molecule comprises a polynucleotide sequence encoding a second HCVR of a bispecific antigen-binding molecule, comprising HCDR1 containing the amino acid sequence of SEQ ID NO: 42, HCDR2 containing the amino acid sequence of SEQ ID NO: 44, and HCDR3 containing the amino acid sequence of SEQ ID NO: 46; A composition in which the third nucleic acid molecule comprises a polynucleotide sequence encoding LCVR, a bispecific antigen-binding molecule, which includes LCDR1 containing the amino acid sequence of SEQ ID NO: 50, LCDR2 containing the amino acid sequence of SEQ ID NO: 52, and LCDR3 containing the amino acid sequence of SEQ ID NO:

54.

45. A composition comprising an isolated bispecific antigen-binding molecule according to any one of claims 1 to 30, an antibody according to claim 31, or a pharmaceutical composition according to claim 32, for inhibiting the growth of plasma cell tumors in a target.

46. The composition according to claim 45, wherein the plasma cell tumor is multiple myeloma.

47. A composition comprising an isolated bispecific antigen-binding molecule according to any one of claims 1 to 30, or an antibody according to claim 31, or a pharmaceutical composition according to claim 32, for inhibiting tumor growth in a target, wherein the tumor is selected from the group consisting of multiple myeloma, B-cell leukemia, hepatocellular carcinoma, non-small cell lung cancer, melanoma, pancreatic ductal adenocarcinoma, glioma, breast cancer, and another cancer partially characterized by having CD38+ cells.

48. The composition according to any one of claims 45 to 47, further comprising a second therapeutic agent.

49. The composition according to claim 48, wherein the second therapeutic agent is an anti-BCMA / anti-CD3 bispecific antigen-binding molecule.

50. The composition according to claim 48, wherein the second therapeutic agent is an anti-CD20 / anti-CD3 bispecific antigen-binding molecule.

51. The composition according to claim 48, wherein the second therapeutic agent comprises a chemotherapeutic agent, a DNA alkylating agent, an immunomodulator, a proteasome inhibitor, a histone deacetylase inhibitor, different bispecific antibodies that interact with different tumor cell surface antigens and T cell or immune cell antigens, an antibody-drug conjugate, a bispecific antibody conjugated to an antitumor agent, PD-1, PD-L1, or CTLA-4 checkpoint inhibitor, or a combination thereof.

52. The composition according to claim 47, wherein the subject is undergoing radiotherapy and / or stem cell transplantation.

53. A composition comprising an isolated bispecific antigen-binding molecule according to any one of claims 1 to 30, an antibody according to claim 31, or a pharmaceutical composition according to claim 32, for treating a patient suffering from multiple myeloma or another BCMA-expressing B-cell malignancy.

54. The composition according to claim 53, wherein the BCMA-expressing B-cell malignancy is selected from the group consisting of Waldenström macroglobulinemia, Burkitt lymphoma, diffuse large B-cell lymphoma, non-Hodgkin lymphoma, chronic lymphocytic leukemia, follicular lymphoma, mantle cell lymphoma, marginal zone lymphoma, lymphoplasmacytic lymphoma, and Hodgkin lymphoma.

55. The composition according to claim 53 or 54, further comprising a second therapeutic agent.

56. The composition according to claim 55, wherein the second therapeutic agent is an anti-BCMA / anti-CD3 bispecific antigen-binding molecule.

57. The composition according to claim 55, wherein the second therapeutic agent comprises a chemotherapeutic agent, a DNA alkylating agent, an immunomodulator, a proteasome inhibitor, a histone deacetylase inhibitor, different bispecific antibodies that interact with different tumor cell surface antigens and T cell or immune cell antigens, an antibody-drug conjugate, a bispecific antibody conjugated to an antitumor agent, PD-1, PD-L1, or CTLA-4 checkpoint inhibitor, or a combination thereof.

58. The composition according to claim 53 or 54, wherein the subject is subjected to radiotherapy and / or stem cell transplantation.

59. A composition comprising, in combination with an anti-PD-1 antibody or its antigen-binding fragment, an isolated bispecific antigen-binding molecule according to any one of claims 1 to 30, or the antibody according to claim 31, or the pharmaceutical composition according to claim 32, for the treatment of patients suffering from CD38+ tumors and / or BCMA-expressing tumors.

60. The composition according to claim 59, wherein the anti-PD-1 antibody is semiprimab.

61. A composition comprising an isolated bispecific antigen-binding molecule according to any one of claims 1 to 30, or an antibody according to claim 31, or a pharmaceutical composition according to claim 32, for the treatment of a disease or disorder related to the expression of CD38 and / or BCMA.

62. The composition according to claim 61, wherein the disease or disorder is cancer.

63. The composition according to claim 62, wherein the cancer is multiple myeloma.

64. The composition according to any one of claims 61 to 63, wherein the antibody, isolated bispecific antigen-binding molecule, or pharmaceutical composition is for further use in combination with an anti-PD-1 antibody or its antigen-binding fragment.

65. The composition according to claim 61, wherein the antibody, isolated bispecific antigen-binding molecule, or pharmaceutical composition is formulated for intravenous, intramuscular, or subcutaneous use.

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