CN114616245A

CN114616245A - anti-CD 38 antibody and application thereof

Info

Publication number: CN114616245A
Application number: CN202080066375.1A
Authority: CN
Inventors: 赵新燕; 邓婧; 李鑫鑫; 卢士强; 赵晓峰; 任晋生
Original assignee: Simcere Biological Medicine Technology Co ltd; Jiangsu Simcere Pharmaceutical Co Ltd
Current assignee: Shandong Simcere Bio Pharmaceutical Co ltd
Priority date: 2019-12-13
Filing date: 2020-12-11
Publication date: 2022-06-10
Anticipated expiration: 2040-12-11
Also published as: WO2021115404A1; CN114616245B

Abstract

The invention discloses an antibody or an antigen binding fragment specifically binding to human CD38, which can mediate tumor cell apoptosis and can be used for treating tumor diseases.

Description

anti-CD 38 antibody and application thereof

The present application claims priority from the chinese patent application filed on 2019, 12/13/h, under the name of "an anti-CD 38 antibody and its use", by the chinese patent office, application No. 201911286272.1, the entire contents of which are incorporated herein by reference.

Technical Field

The present invention relates to anti-human CD38 antibodies or antigen-binding fragments thereof, nucleic acids encoding the same, pharmaceutical compositions comprising the antibodies or antigen-binding fragments, and uses thereof for treating diseases associated with aberrant CD38 expression, such as tumors.

Background

Biological Activity of CD38

CD38 is a type II transmembrane glycoprotein, and binding of CD38 to its ligand, CD31, can affect cell migration and receptor-mediated adhesion functions through interaction with hyaluronic acid. CD38 expression intensity increases after lymphocyte activation, with its expression mainly concentrated in hematopoietic cells; widely present in lymphoid and myeloid cells, but almost absent in most mature resting lymphocytes.

CD38 has various biological effects, belongs to ribose cyclase, and can generate ADP ribose and cyclic ADP ribose by using nicotinamide adenine dinucleotide NAD + as a substrate. This is for extracellular metabolism, intracellular Ca²⁺Cell adhesion and signal transduction, etc. have important regulation and control functions. The natural ligand of CD38 is CD31/PECAM, and the binding of CD38 and CD31 induces tyrosine phosphorylation and downstream signaling to regulate lymphocyte proliferation and cytokine release.

In normal humans, CD38 expression levels are relatively low in bone marrow and lymphocytes, as well as in cells of some non-hematopoietic tissues, compared to high levels of CD38 expression in normal plasma cells and Multiple Myeloma (MM) cells, making CD38 a good target for therapeutic antibody targeting to myeloma cell surface molecules. In addition, CD38 is highly expressed in a variety of cancers, such as prostate cancer, non-small cell lung cancer, multiple myeloma, melanoma, lymphoma, ovarian cancer, breast cancer, and the like. The biological function of CD38 is associated with the regulation of calcium homeostasis of lymphocytes expressing CD 38. Meanwhile, CD38 also has extracellular enzyme activity, and is involved in the generation of nucleotide metabolites and the regulation of intracellular calcium storage.

CD38 monoclonal antibody for treating Multiple Myeloma (MM)

The anti-CD 38 monoclonal antibody is a targeted antibody medicament aiming at a CD38 protein molecule highly expressed on the surface of multiple myeloma cells. The first anti-CD 38 monoclonal antibody Daratumumab binds to CD38 expressed by tumor cells, has antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), and induces programmed cell death (programmed cell death) of tumor cells by cross-linking (cross-linking). Based on its good tolerability and proven exact efficacy, Dratumumab was FDA approved for treatment of relapsed/refractory MM patients in 2015 and was batch marketed in 2019 in 7 months in china. A second CD38 mab, Isatuximab by Sanofi, in clinical stage 3, and pomalidomide in combination with dexamethasone, currently marketed by the FDA and EMA applications, achieved breakthrough in relapsed/refractory MM patients. Because the polypeptide has the function of inhibiting the activity of NDA hydrolase, which can help to inhibit the T cells by inhibiting cells and inhibitory factors in a tumor microenvironment of the antagonistic solid tumor, clinical tests on solid tumors such as liver cancer, head and neck cancer, ovarian cancer, glioblastoma and the like are also developed in the United states at present. The CD38 mAb licensed by Morphosys to Nature, Inc. was reduced from 4-6 hours to about 2 hours at clinical IV infusion compared to Daratumumab and Isatuximab, and may be associated with reduced CDC activity. Clinical phase 3 trials of MM are currently performed in china. We aimed to develop a class of CD38 antibodies that have equal or higher CD38 protein binding affinity and ADCC, ADCP, etc. activity than Daratumumab and Isatuximab, while having little to no CDC activity, but also having the ability to inhibit NDA hydrolase activity.

Relationship between CD38 and drug resistance of immune checkpoint inhibitor drugs such as PD-L1 monoclonal antibody

Clinical data analysis shows that the drug resistance of immune checkpoint inhibitory drugs such as CD38 and PD-L1 antibodies is closely related, and the response rate of patients with high CD38 expression to PD-L1 antibody drugs is strongly related. The relevant mechanism is as follows: 1) the adenosine transdermal path is one of the important mechanisms of drug resistance of drugs such as PD-L1 antibody. After the PD-L1 antibody is administered, high expression of CD38 by tumor cells and Tregs in a tumor microenvironment can be caused, and formation of adenosine is promoted by catalyzing NAD + by CD38, so that T, NK and macrophages are inhibited. Can inhibit adenosine formation by enzyme activity inhibition of CD38 antibody, and resist drug resistance of PD-L1 antibody. 2) The expression of CD38 on immunosuppressive cells such as tumor cells, Tregs and MDSCs of patients with PD-L1 monoclonal antibody drug resistance is obviously up-regulated, CD38 is an important mark of drug resistance of immunosuppressive check points such as PD-L1 antibody and the like, and the immunosuppressive cells in a tumor microenvironment can be reduced through the killing effect of the CD38 monoclonal antibody, so that the tumor killing effect of the immune cells is promoted. At the same time, of course, consideration is also given to how the drug is designed to avoid killing of activated T cells by the CD38 antibody. 3) In addition, the inhibition of the NAD + hydrolase action of the CD38 protein can promote the differentiation of Th0 cells into anti-tumor T cells, inhibit the transformation of Th0 into Treg cells and increase the anti-tumor effect.

Therefore, the development of the CD38 monoclonal antibody which has the effects of killing tumors and inhibiting the activity of NAD hydrolase and can specifically kill the suppressor cells such as Treg, MDSC and the like in the blood tumor cells and the solid tumors with high expression of CD38, or the development of the anti-CD 38X bispecific antibody or the double-target drug containing the antigen binding fragment of the CD38 monoclonal antibody on the basis of the CD38 monoclonal antibody has great social and economic significance for improving the efficacy of treating MM or treating other refractory malignant tumors such as the solid tumors, particularly the solid tumors.

Disclosure of Invention

The invention provides antibodies or antigen-binding fragments that specifically bind to human CD38, nucleic acids encoding these antibodies, nucleic acids comprising the antibodies and antigen-binding fragments and pharmaceutical compositions, and their use for killing tumor cells, for treating tumors. The antibody or antigen binding fragment of the invention can bind not only human CD38 but also cynomolgus monkey CD 38.

In some embodiments, an isolated antibody or antigen-binding fragment that specifically binds human CD38, comprising heavy and light chain CDRs:

(1) the heavy chain CDRs comprise: CDR1-VH, CDR2-VH and CDR 3-VH; the CDRs 1-VH, CDR2-VH and CDR3-VH have any sequence combination selected from the group consisting of or a sequence combination having 1, 2, 3 or more amino acid insertions, deletions and/or substitutions compared to the sequence combination:

and;

(2) the light chain CDRs comprise: CDR1-VL, CDR2-VL and CDR 3-VL; the CDRs 1-VL, CDR2-VL and CDR3-VL have any sequence combination selected from the group consisting of or a sequence combination having 1, 2, 3 or more amino acid insertions, deletions and/or substitutions compared to the sequence combination:

each of the CDRs 1-VH, 2-VH, CDR3-VH, CDR1-VL, CDR2-VL and CDR3-VL is encoded according to the consensus analysis method of KABAT, Chothia or IMGT.

In some embodiments, an isolated antibody or antigen-binding fragment that specifically binds human CD38, comprising a combination of heavy chain CDRs selected from the group consisting of: VH1, VH2, VH3, VH4, VH5, VH6, VH7, VH8, VH9, VH10, VH11, VH12, VH13, VH14, VH15, VH16, VH17, VH18, VH19, VH20, VH21, VH22, VH23, VH24, VH25, VH26, VH27, VH28, VH29, VH30, VH31, VH32, VH33, VH34, VH35, or VH36, and combinations of CDRs having 1, 2, 3, or more amino acid insertions, deletions, and/or substitutions compared to the sequences of the heavy chain CDRs combinations.

In some embodiments, an isolated antibody or antigen-binding fragment that specifically binds human CD38, comprising a combination of light chain CDRs selected from the group consisting of: VL1, VL2, VL3, VL4, VL5, VL6, VL7, VL8, VL9, VL10, VL11, VL12, VL13, VL14, VL15, VL16, VL17, VL18, VL19, VL20, VL21, VL22, VL23, VL24, VL25, VL26, VL27, VL28, VL29, VL30, VL31, VL32, VL33, VL34, VL35, or VL36, and combinations of CDRs having 1, 2, 3, or more amino acid insertions, deletions, and/or substitutions compared to the sequences of the light chain CDRs combinations.

In some embodiments, an isolated antibody or antigen-binding fragment that specifically binds human CD38, comprising a combination of heavy and light chain CDRs selected from the group consisting of: VH + VL, or VH + VL, and combinations of CDRs having 1, 2, 3 or more amino acid insertions, deletions, and/or substitutions compared to the sequence of the heavy and light chain CDRs combination.

In some specific embodiments, an isolated antibody or antigen-binding fragment that specifically binds human CD38, wherein: (1) the heavy chain CDRs comprise:

CDR1-VH comprising SEQ ID NO: 1. 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35 or a CDR1 of VH;

CDR2-VH comprising SEQ ID NO: 1. 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35 or a CDR2 of VH; and the combination of (a) and (b),

CDR3-VH comprising SEQ ID NO: 1. 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35 or a CDR3 of VH; and/or the presence of a gas in the gas,

(2) the light chain CDRs comprise:

CDR1-VL comprising SEQ ID NO: 2. 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 or a CDR1 of a VL as shown in any one of seq id nos;

CDR2-VL comprising SEQ ID NO: 2. 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 or a CDR2 of a VL as shown in any one of seq id nos; and the combination of (a) and (b),

CDR3-VL comprising SEQ ID NO: 2. 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, or a CDR3 of a VL as set forth in any one of

claims

4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36.

In some specific embodiments, an isolated antibody or antigen-binding fragment that specifically binds human CD38, wherein:

(1) the heavy chain variable region and the light chain variable region have sequences shown as SEQ ID NO 1 and SEQ ID NO 2 respectively;

(2) the heavy chain variable region and the light chain variable region have sequences shown in SEQ ID NO 3 and SEQ ID NO 4 respectively;

(3) the heavy chain variable region and the light chain variable region have sequences shown in SEQ ID NO 5 and SEQ ID NO 6 respectively;

(4) the heavy chain variable region and the light chain variable region have sequences shown as SEQ ID NO 7 and SEQ ID NO 8 respectively;

(5) the heavy chain variable region and the light chain variable region have sequences shown in SEQ ID NO 9 and SEQ ID NO 10 respectively;

(6) the heavy chain variable region and the light chain variable region have sequences shown as SEQ ID NO. 11 and SEQ ID NO. 12 respectively;

(7) the heavy chain variable region and the light chain variable region have sequences shown in SEQ ID NO 13 and SEQ ID NO 14, respectively;

(8) the heavy chain variable region and the light chain variable region have sequences shown as SEQ ID NO 15 and SEQ ID NO 16 respectively;

(9) the heavy chain variable region and the light chain variable region have sequences shown in SEQ ID NO 17 and SEQ ID NO 18 respectively;

(10) the heavy chain variable region and the light chain variable region have sequences shown in SEQ ID NO 19 and SEQ ID NO 20, respectively;

(11) the heavy chain variable region and the light chain variable region have sequences shown as SEQ ID NO 21 and SEQ ID NO 22 respectively;

(12) the heavy chain variable region and the light chain variable region have the sequences shown in SEQ ID NO. 23 and SEQ ID NO. 24, respectively;

(13) the heavy chain variable region and the light chain variable region have the sequences shown in SEQ ID NO. 25 and SEQ ID NO. 26, respectively;

(14) the heavy chain variable region and the light chain variable region have the sequences shown in SEQ ID NO 27 and SEQ ID NO 28, respectively;

(15) the heavy chain variable region and the light chain variable region have the sequences shown in SEQ ID NO 29 and SEQ ID NO 30, respectively;

(16) the heavy chain variable region and the light chain variable region have sequences shown as SEQ ID NO 31 and SEQ ID NO 32 respectively;

(17) the heavy chain variable region and the light chain variable region have the sequences shown in SEQ ID NO 33 and SEQ ID NO 34, respectively;

(18) the heavy chain variable region and the light chain variable region have sequences shown in SEQ ID NO 35 and SEQ ID NO 36, respectively; or the like, or, alternatively,

(19) the heavy chain variable region and the light chain variable region each have a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to the sequence shown in (1) to (18) above.

In a preferred embodiment, the antibody or antigen-binding fragment thereof of the invention is:

(1) a chimeric antibody or fragment thereof;

(2) a fully human antibody or fragment thereof; or the like, or, alternatively,

(3) a humanized antibody or fragment thereof.

In a preferred embodiment, the antibody or antigen-binding fragment thereof of the invention binds human CD38 with a dissociation constant (KD) of no greater than 5nM and cynomolgus monkey CD38 with a dissociation constant (KD) of no greater than 25 nM.

In a preferred embodiment, the antibody or antigen-binding fragment thereof of the invention comprises the sequence of the constant region of any one of human or murine antibodies IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, or IgD; preferably comprising the sequence of the constant region of the human or murine antibody IgG1, IgG2, IgG3 or IgG 4.

In a preferred embodiment, the antigen binding fragment of the invention is selected from one or more of f (ab)2, Fab', Fab, Fv, scFv, diabody, nanobody, and antibody minimal recognition unit.

In a preferred embodiment, an antibody or antigen-binding fragment thereof of the invention competes for binding to CD38 or an epitope thereof with an antibody or antigen-binding fragment selected from

numbers

10, 11, 12, 13, 26, 31, 38, 42, 44, 48, 51, 52, 69, 102, 215, 245, 286, or 292 and has the following properties:

1) specifically binds to human CD38 recombinant protein and expresses human CD38 cell;

2) mediates antibody-dependent cellular cytotoxicity killing (ADCC) activity;

3) mediates antibody-dependent cell-mediated phagocytosis (ADCP);

4) (ii) mediating cross-linking induced cell death;

5) no or only weak antibody-mediated complement-dependent cytotoxicity (CDC) activity;

6) inhibiting NAD hydrolase activity; or/and

7) inhibiting tumor growth.

Further, in some embodiments, the antibody or antigen-binding fragment is further conjugated to a therapeutic agent or tracer; preferably, the therapeutic agent is selected from the group consisting of a radioisotope, a cytotoxic agent or an immunomodulatory agent, and the tracer is selected from the group consisting of a radiological contrast agent, a paramagnetic ion, a metal, a fluorescent label, a chemiluminescent label, an ultrasound contrast agent and a photosensitizer; more preferably, the cytotoxic agent is selected from alkaloids (alkaloids), methotrexate (methotrexate), anthracyclines (doxorubicin) or taxanes (taxanes); the toxin compound is preferably DM1, DM4, SN-38, MMAE, MMAF, Duocarmycin, Calichemicin, DX 8951.

Further, in some embodiments, the antibody or antigen-binding fragment is further linked to another functional molecule, which may be selected from one or more of: a signal peptide, protein tag, or cytokine; preferably, the cytokine may be selected from the group consisting of IL-2, IL-6, IL-12, IL-15, IL-21, IFN or TNF-alpha.

In some embodiments, the invention provides an isolated nucleic acid molecule encoding an antibody, antigen-binding fragment, or any combination thereof, of the invention described above.

In some embodiments, the present invention provides an expression vector comprising the isolated nucleic acid molecule of the present invention described above.

In some embodiments, the present invention provides a host cell comprising the isolated nucleic acid molecule or expression vector of the present invention described above.

In a preferred embodiment, the host cell is a eukaryotic cell or a prokaryotic cell; more preferably, the host cell is derived from a mammalian cell, a yeast cell, an insect cell, E.coli and/or Bacillus subtilis; more preferably, the host cell is selected from Chinese hamster ovary Cells (CHO).

In some embodiments, the invention provides a method of producing an antibody or antigen-binding fragment by culturing a host cell of the invention described above under suitable conditions and isolating the antibody or antigen-binding fragment.

In some embodiments, the invention provides a pharmaceutical composition comprising an antibody or antigen-binding fragment of the invention described above, an isolated nucleic acid molecule of the invention described above, an expression vector of the invention described above, a cell of the invention described above, or a product (e.g., an antibody or antigen-binding fragment) made by a method of the invention described above, and a pharmaceutically acceptable carrier.

In a preferred embodiment, the pharmaceutical composition further comprises an additional antineoplastic agent.

In some embodiments, the present invention provides a method of preventing and/or treating a disease associated with aberrant expression of CD38, comprising administering to a patient in need thereof an antibody or antigen-binding fragment of the invention described above, an isolated nucleic acid molecule of the invention described above, an expression vector of the invention described above, a cell of the invention described above, a product (e.g., an antibody and antigen-binding fragment) produced by a method of the invention described above, or a pharmaceutical composition of the invention described above; the disease is preferably a tumor.

In some embodiments, the invention provides the use of an antibody or antigen-binding fragment of the invention described above, an isolated nucleic acid molecule of the invention described above, an expression vector of the invention described above, a cell of the invention described above, a product (e.g., an antibody and antigen-binding fragment) made by a method of the invention described above, or a pharmaceutical composition of the invention described above, in the manufacture of a medicament for the prevention and/or treatment of a disease associated with aberrant expression of CD38, preferably a tumor.

In some embodiments, the invention provides a kit comprising an antibody or antigen-binding fragment of the invention described above, an isolated nucleic acid molecule of the invention described above, an expression vector of the invention described above, a cell of the invention described above, or a product (e.g., an antibody or antigen-binding fragment) made by a method of the invention described above, and instructions for use.

In another aspect, the invention provides a multispecific antibody comprising an antibody or antigen-binding fragment of the first aspect; preferably, the multispecific antibody further comprises an antibody or antigen-binding fragment that specifically binds to an antigen other than CD38 or binds to a different epitope of CD38 than the antibody or antigen-binding fragment of the first aspect.

In some embodiments, preferably, the antigen other than CD38 may be selected from: CD3, preferably CD3 epsilon; CD16, preferably CD 16A; CD 32B; PD-1; PD-2; PD-L1; VEGF; NKG 2D; CD 19; CD 20; CD 40; CD 47; 4-1 BB; CD 137; an EGFR; EGFRvIII; TNF-alpha; CD 33; MSLN; HER 2; HER 3; HAS; CD 5; CD 27; EphA 2; EpCAM; MUC 1; MUC 16; CEA; claudin18.2; a folate receptor; claudin 6; WT 1; NY-ESO-1; MAGE 3; ASGPR1 or CDH 16.

In some embodiments, preferably, the multispecific antibody may be bispecific, trispecific, or tetraspecific, and the multispecific antibody may be bivalent, tetravalent, or hexavalent.

In another aspect, the invention provides a Chimeric Antigen Receptor (CAR) comprising at least an extracellular antigen-binding domain comprising an antibody or antigen-binding fragment selected from any of the antibodies of the invention, a transmembrane domain, and an intracellular signaling domain.

In another aspect, the present invention provides an immune effector cell expressing the chimeric antigen receptor described above, or comprising a nucleic acid fragment encoding the chimeric antigen receptor described above; preferably, the immune effector cell is selected from a T cell, preferably from a cytotoxic, regulatory or helper T cell, a NK cell, a NKT cell, a DNT cell, a double negative T cell, a monocyte, a macrophage, a dendritic cell or a mast cell; preferably, the immune effector cells are autoimmune effector cells or allogeneic immune effector cells.

Terms and definitions:

unless otherwise defined, terms used herein have the meanings that are commonly understood by one of ordinary skill in the art. For a term explicitly defined herein, the meaning of that term shall govern the definition.

The term "CD 38", a glycoprotein, has a cyclic ADP ribohydrolase action and is present on the surface of many immune cells (leukocytes), including T, B lymphocytes and natural killer cells. CD38 also plays a role in cell adhesion, signal transduction, and calcium signaling.

The term "antibody" (Ab) refers to immunoglobulin molecules that specifically bind or are immunoreactive with an antigen of interest, including polyclonal, monoclonal, genetically engineered, and other modified forms of antibodies (including, but not limited to, chimeric, humanized, fully human, heteroconjugate antibodies (e.g., bispecific, trispecific, and tetraspecific antibodies, diabodies, triabodies, and tetrabodies), antibody conjugates), and antigen-binding fragments of antibodies (including, e.g., Fab ', F (Ab') 2, Fab, Fv, rgig, and scFv fragments). Furthermore, unless otherwise indicated, the term "monoclonal antibody" (mAb) is intended to include intact antibody molecules capable of specifically binding to a target protein as well as non-intact antibody fragments (e.g., Fab and F (ab') 2 fragments which lack the Fc fragment of an intact antibody (cleared more rapidly from the animal circulation) and therefore lack Fc-mediated effector function (effector function) (see Wahl et al, J.Nucl.Med.24:316,1983; the contents of which are incorporated herein by reference).

The term "antigen-binding fragment" refers to one or more antibody fragments that retain the ability to specifically bind to a target antigen. The antigen binding function of an antibody may be performed by a fragment of a full-length antibody. The antibody fragment may be a Fab, F (ab') 2, scFv, SMIP, diabody, triabody, affibody, nanobody, aptamer, or domain antibody. Examples of binding fragments encompassing the term "antigen-binding fragment" of an antibody include, but are not limited to: (i) fab fragment, a monovalent fragment consisting of VL, VH, CL and CHl domains; (ii) a f (ab)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bond at the hinge region; (iii) an Fd fragment consisting of the VH and CHl domains; (iv) (ii) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (V) a dAb comprising VH and VL domains; (vi) dAb fragments consisting of VH domains (Ward et al, Nature341:544-546, 1989); (vii) a dAb consisting of a VH or VL domain; (viii) an isolated Complementarity Determining Region (CDR); and (ix) a combination of two or more isolated CDRs, which may optionally be joined by a synthetic linker. Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, these two domains can be joined using recombinant methods by a linker that enables them to be made into a single protein chain in which the VL and VH regions pair to form a monovalent molecule (known as single chain Fv (scFv); see, e.g., Bird et al, Science 242: 423-. These antibody fragments can be obtained using conventional techniques known to those skilled in the art, and these fragments are screened for use in the same manner as intact antibodies. Antigen-binding fragments may be produced by recombinant DNA techniques, enzymatic or chemical cleavage of intact immunoglobulins, or in some embodiments by chemical peptide synthesis procedures known in the art.

The term "bispecific antibody" refers to an antibody, typically a human or humanized antibody, having monoclonal binding specificity for at least two different antigens. In the present invention, one of the binding specificities can be detected against an epitope of CD38, the other against another epitope of CD38 or any other antigen, e.g. against a cell surface protein, receptor subunit, tissue specific antigen, virus derived protein, virus encoded envelope protein, bacteria derived protein or bacteria surface protein etc.

The term "chimeric" antibody refers to an antibody having the variable sequences of an immunoglobulin derived from one organism of origin (e.g., rat or mouse) and the constant regions of an immunoglobulin derived from a different organism (e.g., human). Methods for producing chimeric antibodies are known in the art. See, e.g., Morrison,1985, Science 229(4719): 1202-7; oi et al, 1986, Bio Techniques 4: 214-; gillies et al, 1985J Immunol Methods 125: 191-202; the above is incorporated by reference herein.

The term "humanized antibody" refers to an antibody obtained by modifying a murine monoclonal antibody by gene cloning and DNA recombination technology and re-expressing, wherein most of the amino acid sequences of the antibody are replaced by human sequences, so that the affinity and specificity of the parent murine monoclonal antibody are basically maintained, the heterogeneity is reduced, and the antibody is favorably applied to human bodies. A humanized antibody is one in which the constant region portion (i.e., the CH and CL regions) of the antibody or all of the antibody is encoded by the human antibody gene. Humanized antibodies can greatly reduce the immunological side effects of heterologous antibodies on the human body.

The term "complementarity determining region" (CDR) refers to a hypervariable region found in both the light chain and heavy chain variable domains. The more highly conserved portions of the variable domains are called the Framework Regions (FR). As understood in the art, the amino acid positions representing hypervariable regions of an antibody can vary according to the context and various definitions known in the art. Some positions within a variable domain may be considered to be hybrid hypervariable positions in that these positions may be considered to be within a hypervariable region under one set of criteria (such as IMGT or KABAT) but outside a hypervariable region under a different set of criteria (such as KABAT or IMGT). One or more of these positions may also be found in extended hypervariable regions. The invention includes antibodies comprising modifications in these hybrid hypervariable positions. The variable domains of native heavy and light chains each comprise four framework regions that are predominantly in a sheet configuration, connected by three CDRs (CDR1, CDR2, and CDR3) that form loops that connect, and in some cases form part of, the sheet structure. The CDRs in each chain are held tightly together by the FR regions in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and contribute to the formation of the antigen binding site of the antibody with the CDRs from the other antibody chain (see Kabat et al, Sequences of Protein of immunological Interest, National Institute of Health, Bethesda, Md. 1987; which is incorporated herein by reference). For example, herein, CDRs 1-VH, CDR2-VH and CDR3-VH refer to the first CDR, second CDR and third CDR, respectively, of a heavy chain variable region (VH), which constitute the CDR combination (VHCDR combination) of the heavy chain (or its variable region); CDRs 1-VL, CDRs 2-VL and CDRs 3-VL refer to the first CDR, second CDR and third CDR, respectively, of the light chain variable region (VL), which constitute the combination of CDRs (VLCDR combination) of the light chain (or its variable region).

The term "VH" refers to the variable region of the immunoglobulin heavy chain of an antibody (including the heavy chain of Fv, scFv or Fab). The term "VL" refers to the variable region of an immunoglobulin light chain (including the light chain of an Fv, scFv, dsFv, or Fab).

The term "antibody conjugate" refers to a conjugate formed by an antibody molecule chemically bonded to another molecule, either directly or through a linking linker. Such as an antibody-drug conjugate (ADC) in which the drug molecule is the other molecule.

The term "monoclonal antibody" refers to an antibody derived from a single clone (including any eukaryotic, prokaryotic, or phage clone) and is not limited to the method of production of the antibody.

The term "percent (%) sequence identity" refers to the percentage of amino acid (or nucleotide) residues of a candidate sequence that are identical to the amino acid (or nucleotide) residues of a reference sequence, after aligning the sequences and introducing gaps, if necessary, for maximum percent sequence identity (e.g., gaps can be introduced in one or both of the candidate and reference sequences for optimal alignment, and non-homologous sequences can be omitted for comparison purposes). Alignment can be achieved in a variety of ways well known to those skilled in the art for the purpose of determining percent sequence identity, for example using publicly available computer software such as BLAST, ALIGN or Megalign (DNAsari) software. One skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm that requires maximum alignment over the full length of the sequences being compared. For example, a reference sequence aligned for comparison to a candidate sequence can show that the candidate sequence exhibits from 50% to 100% sequence identity over the full length of the candidate sequence or over a selected portion of contiguous amino acid (or nucleotide) residues of the candidate sequence. The length of a candidate sequence aligned for comparison purposes can be, e.g., at least 30% (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) of the length of the reference sequence. When a position in the candidate sequence is occupied by the same amino acid (or nucleotide) residue as the corresponding position in the reference sequence, then the molecules are identical at that position.

The term "specific binding" refers to a binding reaction that determines the presence of an antigen in a heterogeneous population of proteins and other biomolecules that are specifically recognized, for example, by antibodies or antigen-binding fragments thereof. An antibody or antigen-binding fragment thereof that specifically binds to an antigen will bind to the antigen with a KD of less than 100 nM. For example, an antibody or antigen-binding fragment thereof that specifically binds to an antigen will bind to the antigen with a KD of up to 100nM (e.g., between 1pM and 100 nM). An antibody or antigen-binding fragment thereof that does not exhibit specific binding to a particular antigen or epitope thereof will exhibit a KD for that particular antigen or epitope thereof of greater than 100nM (e.g., greater than 500nM, 1 μ Μ, 100 μ Μ, 500 μ Μ or 1 mM). A variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein or carbohydrate. For example, solid phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein or carbohydrate. See, Harlow & Lane, Antibodies, organic Manual, Cold Spring Harbor Press, New York (1988) and Harlow & Lane, Using Antibodies, A Laboratory Manual, Cold Spring Harbor Press, New York (1999), which describe immunoassay formats and conditions that can be used to determine specific immunoreactivity.

The term "Chimeric Antigen Receptor (CAR)" refers to an artificial cell surface receptor engineered to express on an immune effector cell and specifically bind an antigen, which comprises at least (1) an extracellular antigen-binding domain, such as the variable heavy or light chain of an antibody, (2) a transmembrane domain that anchors the CAR into the immune effector cell, and (3) an intracellular signaling domain. CARs are capable of utilizing extracellular antigen-binding domains to redirect T cells and other immune effector cells to a selected target, such as cancer cells, in a non-MHC-restricted manner.

The term "ADCC" (antibody-dependent cell-mediated cytotoxicity, ADCC), which is an antibody-dependent cell-mediated cytotoxicity, refers to the Fab fragment of an antibody that specifically recognizes an epitope of a tumor cell, and the Fc fragment thereof binds to an FcR on the surface of a killer cell (NK cell, macrophage, etc.) to mediate direct killing of the killer cell against a target cell. The ADCC mediated by the antibody is strong and weak and is related to a plurality of factors, such as the affinity of the antibody and an antigen, the affinity of the antibody and an Fc fragment receptor, the characteristics of immune effector cells and the like. In general, antibodies with high affinity for antigens or Fc receptors mediate stronger ADCC effects. The glycosylation and the amino acid sequence modification of the Fc segment of the antibody can improve the ADCC activity. In the glycosylation modification of an antibody, fucose is considered to be the most important sugar affecting ADCC activity, and defucosylation can significantly improve the affinity of the antibody to Fc γ RIIIa and ADCC activity.

The term "antibody induces programmed cell death by cross-linking" (antibody induced cell death-linking) refers to the induction of a programmed cell death process by the Fc fragment of an antibody with an Fc receptor (FcR) or after the action of a second cross-linking antibody. ADCC and ADCP are the induction of aggregated IgG constant domains (Fc domains) by monoclonal antibody binding to Fc γ Rs on activated immune effector cells such as natural killer cells, macrophages and polymorphonuclear cells. Monoclonal antibodies can interact with Fc receptors via the Fc fragment of the antibody to enhance the agonistic activity of the antibody or induce Programmed Cell Death (PCD). Agonist therapeutic monoclonal antibodies targeting death receptor family members induce PCD through an extrinsic apoptotic pathway. Agonistic monoclonal antibody-induced PCD targeting these death receptors is enhanced by crosslinking, such as by secondary crosslinking antibodies or more physiologically binding to Fc γ Rs. Antibody-mediated antigen crosslinking, unrelated to the death receptor family, can also induce PCD, but not through the classical apoptotic pathway. This pathway is characterized by homotypic aggregation of cells, including cytoskeletal reorganization, lysosomal activation, and reactive oxygen species production. This non-inducible PCD pathway may be enhanced by Fc cross-linked secondary antibodies or Fc γ R expressing cells.

The term "vector" includes nucleic acid vectors, such as DNA vectors (e.g., plasmids), RNA vectors, viruses, or other suitable replicons (e.g., viral vectors). Various vectors have been developed for the delivery of polynucleotides encoding foreign proteins into prokaryotic or eukaryotic cells. The expression vectors of the invention contain polynucleotide sequences as well as additional sequence elements, e.g., for expressing proteins and/or integrating these polynucleotide sequences into the genome of mammalian cells. Some vectors that may be used to express the antibodies and antibody fragments of the invention include plasmids containing regulatory sequences (e.g., promoter and enhancer regions) that direct the transcription of genes. Other useful vectors for expressing antibodies and antibody fragments contain polynucleotide sequences that enhance the rate of translation of these genes or improve the stability or nuclear export of mRNA produced by transcription of the genes. These sequence elements include, for example, 5 'and 3' untranslated regions, Internal Ribosome Entry Sites (IRES), and polyadenylation signal sites to direct the efficient transcription of genes carried on expression vectors. The expression vectors of the invention may also contain a polynucleotide encoding a marker for selecting cells containing such a vector. Examples of suitable markers include genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, kanamycin or nourseothricin.

The terms "subject", "subject" and "patient" refer to an organism that is receiving treatment for a particular disease or disorder as described herein, such as cancer or an infectious disease. Examples of subjects and patients include mammals, such as humans, primates, pigs, goats, rabbits, hamsters, cats, dogs, guinea pigs, members of the family bovidae (e.g., cattle, bison, buffalo, elk, and yak, etc.), cattle, sheep, horses, and bison, etc., that are treated for a disease or disorder (e.g., a cell proliferative disorder, such as cancer or an infectious disease).

The term "treatment" refers to surgery or drug treatment (therapeutic or therapeutic treatment) for the purpose of preventing, slowing (reducing) the progression of an undesirable physiological change or disorder, such as a cell proliferative disorder (e.g., cancer or infectious disease), in a subject being treated. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or complete), whether detectable or undetectable. Subjects in need of treatment include subjects already suffering from a condition or disease as well as subjects susceptible to a condition or disease or subjects for whom prevention of a condition or disease is intended. When terms such as slow, etc., are referred to, their meanings also include the elimination, disappearance, absence, etc.

The term "effective amount" refers to an amount of a therapeutic agent that is effective to prevent or ameliorate a disease condition or the progression of the disease when administered alone or in combination with another therapeutic agent to a cell, tissue, or subject. An "effective amount" also refers to an amount of a compound that is sufficient to alleviate symptoms, e.g., to treat, cure, prevent or alleviate the associated medical condition, or to increase the rate at which such condition is treated, cured, prevented or alleviated. When the active ingredient is administered alone to an individual, a therapeutically effective dose refers to the ingredient alone. When a combination is used, a therapeutically effective dose refers to the combined amounts of the active ingredients that produce the therapeutic effect, whether administered in combination, sequentially or simultaneously.

The term "cancer" refers to or describes a physiological condition in mammals that is typically characterized by unregulated cell growth. Included in this definition are benign and malignant cancers. As used herein, the term "tumor" or "neoplasm" refers to all neoplastic (neoplastic) cell growth and proliferation, whether malignant or benign, and all pre-cancerous (pre-cancer) and cancerous cells and tissues. The terms "cancer" and "tumor" are not mutually exclusive when referred to herein.

Drawings

The foregoing and other aspects of the invention will become apparent from the following detailed description of the invention and the accompanying drawings. The drawings herein are for the purpose of illustrating certain preferred embodiments of the invention, it being understood, however, that the invention is not limited to the specific embodiments disclosed.

FIG. 1A, CD38-His immune mouse serum was tested for binding titer to human CD 38-His;

FIG. 1B, CD38-His immunized mouse sera tested for binding titer to human EGFR-His;

FIG. 2A, result of CD38-His staining of spleen B cells of non-immunized mice;

FIG. 2B, CD38-His immunized mice spleen B cell CREG-His staining results;

FIG. 2C, CD38-His immunized mice splenic B cells CD38-His staining results;

figure 3A, FACS detects direct binding EC50 values of anti-CD 38 candidate antibodies and Daudi cells;

FIG. 3B, FACS detects direct binding of candidate antibodies to CHO-CD38 cells to EC50 values;

FIGS. 4A-4B, antibody dependent cell-mediated phagocytosis (ADCP) assay of anti-CD 38 antibody;

FIG. 5A, phosphatidylserine translocation of anti-CD 38 antibody to Romas cells induced by cross-linking;

FIG. 5B, cell death induced by cross-linking of anti-CD 38 antibody;

FIG. 6A, dose dependence of Annexin V positive cells induced by cross-linking of anti-CD 38 antibody, at a higher ratio than PI positive cells;

FIG. 6B dose-dependent relationship of anti-CD 38 antibody induced cell death by cross-linking;

FIGS. 7A-7B, ADCC Activity assay of NK cells on Daudi cells mediated by anti-CD 38 antibody;

FIGS. 8A-8C, ADCC Activity assay of PBMCs on Daudi cells mediated by anti-CD 38 antibody;

FIG. 9 detection of inhibition of NAD hydrolase activity of CD38 protein by anti-CD 38 antibody;

FIGS. 10A-10B, complement dependent cytotoxicity response mediated by anti-CD 38 antibody.

Detailed Description

The present invention is described in detail below with reference to examples and the attached drawings, which are intended to illustrate some preferred embodiments of the invention, however, it is to be understood that the invention is not limited to the specific embodiments disclosed or is to be considered as limiting the scope of the invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

Example 1 immunization of mice produces monoclonal antibodies that specifically bind to CD38

Female SJL mice (purchased from Beijing Wintonli laboratory animal technologies, Inc.) or Balb/c mice (purchased from Shanghai Si Rick laboratory animals, Inc.) at 6-8 weeks of age were immunized for the first time with human CD38-His (Novoprotein, Cat: CU65) and Freund's complete adjuvant (complete free's adjuvant, Sigma, Cat: F5881); three post-immunizations were performed using human CD38-His as described above with incomplete Freund's adjuvant (IFA, Sigma, Cat: F5506) plus unmethylated cytosine guanine dinucleotide (CpGODN1826, synthesized from Shanghai Probiotics); when the vaccine is used, 50 mu g of the emulsion is injected per time, and the emulsion is formed uniformly and stably through an emulsification operation. In particular, the foot pad and the back are injected after the first and second immunization injection, and the tail part is injected subcutaneously and the back are injected for the third and fourth immunization injection, so that antiserum with high titer, high affinity and high specificity and specific immune cells are obtained. On day 5-7 after the last immunization (fourth immunization), mice were euthanized and spleens were aseptically removed, splenic lymphocytes of the mice were aseptically isolated and extracted, and were split into cryopreserved tubes, which were cryopreserved in liquid nitrogen. Blood collection was performed on the mice at the second immunization, 10 days after the third immunization, and the day of euthanasia, respectively, and the sera were separated and the titer of anti-CD 38-specific antibody in the sera was determined by enzyme-linked immunosorbent assay (ELISA).

The experimental results are shown in FIGS. 1A and 1B, and show that the serum of the immunized mouse can be combined with human CD38-His with high titer after four times of immunization. And the binding titer with the His-unrelated protein (human EGFR-His) is low, which indicates that the method can be used for immunizing mice and can ensure that the mice can generate anti-CD 38 antibody with high titer and high specificity.

Example 2 flow cytofluorescent sorting (FACS) of CD 38-specific Single B cells

Spleen cells of mice immunized with CD38 protein, antigen CD38-His protein (CD38-His, Novoprotein, Cat: CU65) and indirect labeled antibody anti-His-APC (R)&D Systems, Cat. IC050A) and antibodies against a marker specific for mouse B cells (anti-mouse B220-Pacfic Blue, BD Biosciences, Cat.558108; anti-mouse IgD-PE, BD Biosciences, Cat.558597; anti-mouse IgM-PE cy7, BD Biosciences, Cat.552867), and a dye 7-AAD (BD Biosciences, Cat.51-68981E) for distinguishing dead cells from live cells was added before sorting, and individual B cells (7 AAD) specific to CD38 were sorted using an AriaIII (BD Co.) flow cytometer^-B220 ⁺IgD ^-IgM ^-CD38 ⁺) One cell was collected per well into a PCR well containing cell lysate, rnase inhibitor. The results showed that spleen of mice immunized with the CD38-His protein (FIG. 2A) or with the CD38 protein stained with the unrelated protein CREG-His (FIG. 2B) did not detect CD38⁺B cells, while spleen from CD38-His immunized mice (FIG. 2C) detected a significant population of CD38⁺B cells, 10 times⁶About 65 CD38 cells in each spleen cell⁺B cells.

Example 3 amplification and high throughput expression of monoclonal antibodies

The following patents "a composite primer for nested amplification and its applications" patent application No.: 201811618134.4 the method of example 1, reverse transcribing mRNA from a single cell into cDNA. Then, nested PCR was performed using cDNA as a template, and amplification of heavy and light chains of the antibody was performed, respectively. Amplifying to obtain the heavy chain variable region and the light chain variable region of the antibody, and cloning to a heavy chain expression vector and a light chain expression vector respectively by a homologous recombination method. The constant regions of both the heavy chain and light chain expression vectors were derived from human IgG 1. The complete heavy chain expression sequence is the signal peptide-VH-CH 1-hinge region-CH 2-CH3 and the complete light chain expression sequence is the signal peptide-Vkappa-Ckappa. The single B cell antibody cloning and expression described above achieves rapid identification and discovery of antibodies in a high throughput manner in 96-well plates. 18 positive candidate antibody molecules are obtained after the cloned and expressed antibody heavy chain and light chain are subjected to a series of physicochemical and functional screening 324, CDRs of sequences of the 18 positive candidate antibody molecules are analyzed by IMGT and KABAT software respectively, corresponding sequence information is shown in the following table 1, wherein the table 1 shows VH and VL sequences of the candidate antibody molecules, and the table 2 shows IMGT and KABAT analysis results of the candidate antibody molecules).

The candidate anti-CD 38 antibody was sequenced and the specific sequence information for the heavy chain variable region and the light chain variable region was as follows:

TABLE 1 detailed sequence information of heavy chain variable region and light chain variable region of anti-CD 38 antibody

The CDRs of each antibody were analyzed using IMGT and KABAT software, respectively, and the specific sequence information is as follows:

TABLE 2 analysis of the CDRs specific sequence information of each antibody by IMGT and KABAT software

Example 4 Octet detection of antibody binding affinity and specificity

Using an Octet HTX detection system, antibodies were diluted to 1. mu.g/ml with PBST, antigen CD38-His (Novoprotein, Cat: CU65) was diluted to 100nM with PBST as the starting concentration, 2-fold gradient dilution, 7 gradients were added sequentially to a 96-well Octet plate (Greiner, Cat.655209), 300. mu.l/well; setting a circulation program, wherein 8 protein A probes are arranged in total, each circulation loads the antibody to the height of 1nm in about 3 minutes, then the probe is combined with the CD38-His antigen after gradient dilution in about 10 minutes to a saturated platform, and finally the regeneration is carried out by using Glycine pH1.5. The experimental data were analyzed and the equilibrium dissociation constant KD of the antibody antigen was fitted to determine the association rate constant ka and the dissociation rate constant KD.

The affinity of antibody candidates for human and cynomolgus monkey CD38 protein is summarized in table 3. The association rate Kon, the dissociation rate Kdis, the dissociation constant KD. It can be seen from table 3 that the candidate CD38 antibody has a much improved binding affinity to human as compared to the commercially available drug control antibody Daratumumab (Janssen, cat. gis0503), but has a weaker binding affinity to cynomolgus monkey than Daratumumab, and has a substantially equivalent or improved binding affinity to human and cynomolgus monkey as compared to the CD38 mab positive control Mor202-kaps (Biointron, cat. b4163) prepared according to the published sequences of the Morphosys patent.

TABLE 3 Octet detection of CD38 antibody binding affinity and specificity

Example 5 determination of EC50 of candidate antibodies by flow cytometry (FACS)

The dose-dependent binding ability of the candidate anti-CD 38 antibody to the CD38 target on the cell surface was confirmed by flow cytometry (FACS).

2 cell strains with high expression of CD38 were selected for this experiment: daudi (purchased from cell Bank of Chinese academy of sciences, Cat: TChu140) and CHO-38 cells (ordered from Yongshan Biotechnology, Inc.) with high expression of CD38 protein on CHO cell surface.In U-shaped round-bottom 96-well plates, 1X 10⁵Daudi or CHO-38 cells and serial dilutions of anti-CD 38 antibody starting at 10. mu.g/ml, 10-fold gradients of 6 per antibody, were incubated for half an hour at 4 ℃ and then 5. mu.g/ml of a secondary anti hIgG Fc (Invitrogen, Cat:31125) was added and the machine FACS detection was performed after half an incubation at 4 ℃.

The results demonstrate that the candidate antibody efficiently binds to the cell surface expressing CD38, as shown in fig. 3, the reference antibody Daratumumab (positive control) has an EC50 of 57.94ng/ml on the Daudi cell surface (fig. 3A) and an EC50 of 60.43ng/ml on the CHO-38 cell surface (fig. 3B), whereas the EC50 of the candidate antibody binding to CD38 on both cell surfaces is not much different from the reference antibody; when the reference antibody was Mor202-kaps (positive control), the EC50 for the candidate antibody was lower on both cells than the reference antibody, indicating that the candidate antibody bound more strongly to both cells than to Mor202-kaps, as detailed in Table 4.

TABLE 4 EC50 result chart

Antibody numbering	Daudi-EC50(ng/ml)	CHO-38-EC50(ng/ml)
11	58.36	111.9
13	57.97	123.1
42	128	89.55
44	57.47	116.5
48	86.47	99.59
69	131	67.41
102	58.23	86.21
215	82.71	106.8
286	149.8	109.8
292	94.59	61.08
Daratumumab	57.94	60.43
Mor202-Kaps	322.1	178.2

Example 6 antibody-dependent cell-mediated phagocytosis (ADCP) assay of anti-CD 38 antibody

ADCP was measured using Daudi cells (purchased from cell Bank of China academy of sciences, Cat: TCTU 140) as target cells and human macrophages (induced by monocyte isolation from PBMC) as effector cells.

Preparation of effector cells: peripheral blood mononuclear cells PBMC (Stemexpress, Cat: PB0004C) from frozen normal humans were incubated overnight in RPMI1640(Gibco, Cat: 22400-. Thereafter, the cells were collected and then subjected to viable cell counting. Then, monocytes were isolated from PBMC by inducing differentiation of monocytes in RPMI1640 containing 10% FBS (Gibco, Cat:10099-141) supplemented with 100. mu.g/ml GM-CSF (Peprotech, Cat:300-03-20Ug) and 5% CO, with specific reference to the protocol of the monocyte isolation kit (EasySep Cat:19058)₂After one week of culture at 37 ℃ in culture, the cells were counted after resuspension and used as effector cells.

Preparation of target cells: target cells Daudi (purchased from cell Bank of Chinese academy of sciences, catalog number: TCTU 140) were collected and labeled with CelltraceTMviolet (Invitrogen, Cat: C34556), and the labeled cells were resuspended in RPMI1640 supplemented with 10% FBS and used as target cells.

In U-shaped round-bottom 96-well plates, 5X 10⁴Daudi cells and diluted anti-CD 38 antibody at a final concentration of 1. mu.g/ml, 0.1. mu.g/ml, preincubated at 4 ℃ for half an hour, then 2X 10 added⁵Effector cells (effector cells: target cells ═ 4: 1) were incubated at 4 ℃ for 3 hours. After the incubation is completed, a staining operation is performed.

And (3) dyeing: after blocking Human TruStain FcX (Biolegend, Cat:422302) for half an hour, staining antibodies APC Mouse anti Human CD19(BD, Cat:555415), AF488Mouse anti Human CD11b (BD, Cat:557701) were added and incubated at 4 ℃ for half an hour. The suspension was resuspended after two washes with DPBS (Hyclone, Cat: SH30256.01) containing 2% FBS (Gibco, Cat:10099-141), and PI (Invitrogen, Cat: P3566) was added before loading onto the machine for analysis by flow cytometry.

The cell lysis rate caused by ADCP activity was calculated according to the following calculation formula:

rate of phagocytosis by cellsCell proportion of (4) (CD 19)⁺CD11b ^-) Percentage of target cells (Celltrace)⁺)*100％

Referring to FIGS. 4A and 4B, it can be seen that both the candidate anti-CD 38 antibody enhanced ADCP effect, and that candidate antibody 69 had a greater ADCP effect at both doses of 1 μ g/ml and 0.1 μ g/ml than the reference antibody Daratumumab (positive control).

Example 7 analysis of programmed cell death induced by Cross-linking of anti-CD 38 antibodies

Apoptosis induced by CD38 antibody cross-linking was determined on Romas cells (Shanghai enzyme research Biotech Co., Ltd., cat. CC-Y1430). In U-shaped round-bottom 96-well plates, 4X 10⁴Romas cells and serial dilutions of anti-CD 38 antibody at final concentrations of 10. mu.g/ml, 1. mu.g/ml, 0.1. mu.g/ml and 0.01. mu.g/ml were preincubated for half an hour, followed by addition of 5. mu.g/ml anti hIgG Fc (Invitrogen, Cat:31125) at 5% CO₂And cultured at 37 ℃ for 20 hours. Cells were stained with Annexin V and PI in the apoptosis detection kit (eBioscience, Cat: BMS500FI/300), respectively, and analyzed by flow cytometry. The percent Annexin V positive and the percent PI positive were computationally analyzed using Flowjo software.

The result shows that all candidate CD38 antibodies (0.1 mu g/ml) can induce phosphatidylserine translocation of Romas cells under the action of Fc cross-linked secondary antibody anti hIgGFc (FIG. 5A), so that the Annexin V positive proportion of Romas cells is obviously improved. The positive proportion of PI cells was also significantly increased, i.e. cells were dead (FIG. 5B). The proportion of Annexin V positive cells is higher than that of PI positive cells, the influence of CD38 antibodies with different concentrations on the cells is further observed (figures 6A-6B), and the candidate anti-CD 38 antibodies can effectively induce the apoptosis of the cells in the presence of an Fc crosslinking reagent at the concentration of 0.01-10 mu g/ml.

Example 8 ADCC Activity assay for anti-CD 38 antibodies

Preparation of effector cells: peripheral blood mononuclear cells PBMC (Stemexpress, Cat: PB0004C) from cryopreserved normal humans were incubated overnight in RPMI1640(Gibco, Cat: 22400-. Thereafter, the cells were collected and then subjected to viable cell counting. Then, NK cells were isolated and purified from the PBMC, and NK cells (Stemcell Cat:17955) were isolated with specific reference to the NK cell isolation kit instructions, and counted after being resuspended in RPMI1640 supplemented with 10% FBS (Gibco, Cat:10099-141) and used as effector cells. PBMCs can be used directly for effector cells of ADCC experiments.

The anti-CD 38 antibody was diluted with RPMI1640 supplemented with 10% FBS, the diluted antibody was dispensed into U-bottom 96-well plates at 50 μ l/well, and then labeled target cells were added to the wells. The plate was heated at 37 ℃ with 5% CO₂Preincubation was carried out for half an hour in an incubator. Subsequently, effector cells were added to the wells (effective target ratio NK: Daudi 2:1-4: 1; PBMC: Daudi 20:1-40:1), 37 ℃, 5% CO₂Incubate in incubator for 3-4 hours. The plate was removed and the fraction of dead cells in the CelltraceTMviolet positive target cells, i.e., PI positive, was measured by flow analysis after adding the dead cell marker dye PI (1. mu.l/well) to each well. The cell lysis rate caused by ADCC activity was calculated according to the following calculation formula:

cell lysis (%) - (% sample well PI% -target cell well only PI%)/(1-target cell well only PI%).

The results are shown in FIGS. 7A-7B and FIGS. 8A-8C, all of the anti-CD 38 antibodies showed lytic killing activity on Daudi cells in an antibody concentration-dependent manner. The EC50 for ADCC activity of CD38 antibody was between 0.008-0.029ng/ml with NK as effector cells (FIGS. 7A-7B), and EC50 for ADCC activity of CD38 antibody was between 0.084-0.200ng/ml with PBMC as effector cells (FIGS. 8A-8C). The ADCC activity of the positive control Daratumumab (Dala) was stronger than that of Mor202-kaps, and the negative control antibody anti-Hel (manufactured by Producer organisms) (no binding to Daudi cells) was consistent with expectations, with no ADCC activity.

Example 9 detection of inhibition of the protease Activity of CD38 by the CD38 antibody

This example uses a screening kit for inhibitors of the activity of CD38 hydrolase (BPS Bicoscience, Cat.79287) and is carried out according to the instructions. Adding a hydrolysis buffer solution, 1 mu g/ml of an antibody to be detected and 0.5 mu g/ml of CD38 protein into a 96-well plate, putting the system into an incubator at 37 ℃ for incubation for 30 minutes, then adding an epsilon-NAD substrate, carrying out fluorescence detection by an enzyme-linked immunosorbent assay instrument, wherein the excitation wavelength is 300nm, the detection wavelength is 410nm, reading the fluorescence value of each sample hole on the plate every 3 minutes, and reading for 60 minutes. Selecting a previous time point entering a plateau period according to a time-fluorescence value curve, and calculating an inhibition rate:

the inhibition ratio (%) - (Sample fluorescence value-Blank fluorescence value)/(Positive Control fluorescence value-Blank fluorescence value) × 100%.

As shown in FIG. 9, the control Drug Daratumumab (Dara) showed no inhibition of CD38 hydrolase activity, and the reaction curves of the negative control antibody group IgG1 kappa and the Drug-free group Drug0 coincided with each other; the response curve of the control drug Isatuximab (Isa) with the function of inhibiting the CD38 hydrolase activity is similar to that of the anti-CD 38

antibody

102 and 215, and the response rate of the CD38 hydrolase activity is obviously reduced, which shows that the tested anti-CD 38 antibody can effectively inhibit the hydrolase activity of the CD 38.

Example 10 CDC Activity assay of CD38 antibody

The following cells and antibodies were added to 96-well plates: daudi cell 1X 10⁵cell/well, 25 μ l/well, CD38 antibody: 25 μ l/well, 4 μ g/ml starting concentration, 1:10 dilution, and incubation at 37 ℃ for 30 min. Human serum (human serum for first sound medical examination) was added to make the final concentration 5%. Incubate at 37 ℃ for 2.5 hours. The PI dye (invitrogen, Lot:1887160) was diluted 1:20 with RPMI1640, added 20. mu.l/well to give a final PI dose of 1. mu.l/well, incubated for 5 minutes at room temperature, and then detected by flow cytometry to record the proportion of dead cells. CDC activity (%) ═ PI positive cell fraction. The results are shown in FIGS. 10A-10B, where Daratumumab, known to have strong CDC activity, showed dose-dependent CDC activity as expected, whereas the CD38 candidate antibodies tested had insignificant CDC activity except for

antibodies

10, 31, 38, 102, 215 at the highest three concentrationsExhibiting CDC activity. These antibodies are expected to avoid an infusion-related reaction (IRR) due to CDC activity.

Claims

An isolated antibody or antigen-binding fragment that specifically binds human CD38, wherein the antibody or antigen-binding fragment comprises heavy and light chain CDRs:

(1) the heavy chain CDRs comprise: CDR1-VH, CDR2-VH and CDR 3-VH; the CDRs 1-VH, CDR2-VH and CDR3-VH have any sequence combination or sequence combination with 1, 2, 3 or more amino acid insertions, deletions and/or substitutions compared to the sequence combination selected from:

and;

(2) the light chain CDRs comprise: CDR1-VL, CDR2-VL and CDR 3-VL; the CDRs 1-VL, CDR2-VL, and CDR3-VL have any sequence combination selected from the group consisting of or a combination of sequences having 1, 2, 3, or more amino acid insertions, deletions, and/or substitutions as compared to the sequence combination:
the antibody or antigen-binding fragment thereof of claim 1, comprising a combination of heavy chain CDRs selected from the group consisting of: VH1, VH2, VH3, VH4, VH5, VH6, VH7, VH8, VH9, VH10, VH11, VH12, VH13, VH14, VH15, VH16, VH17, VH18, VH19, VH20, VH21, VH22, VH23, VH24, VH25, VH26, VH27, VH28, VH29, VH30, VH31, VH32, VH33, VH34, VH35, or VH36, and combinations of CDRs having 1, 2, 3, or more amino acid insertions, deletions, and/or substitutions compared to the sequences of the heavy chain CDRs combinations.
The antibody or antigen-binding fragment thereof of claim 1, comprising a combination of light chain CDRs selected from the group consisting of: VL1, VL2, VL3, VL4, VL5, VL6, VL7, VL8, VL9, VL10, VL11, VL12, VL13, VL14, VL15, VL16, VL17, VL18, VL19, VL20, VL21, VL22, VL23, VL24, VL25, VL26, VL27, VL28, VL29, VL30, VL31, VL32, VL33, VL34, VL35, or VL36, and combinations of CDRs having 1, 2, 3, or more amino acid insertions, deletions, and/or substitutions compared to the sequences of the light chain CDRs combinations.
The antibody or antigen binding fragment thereof of any one of claims 1-3, comprising a combination of heavy and light chain CDRs selected from the group consisting of: VH + VL, or VH + VL, and combinations of CDRs having 1, 2, 3 or more amino acid insertions, deletions, and/or substitutions compared to the sequence of the heavy and light chain CDRs combination.
The antibody or antigen-binding fragment of any one of claims 1 to 4, characterized in that,

(1) the heavy chain CDRs comprise:

CDR1-VH comprising SEQ ID NO: 1. 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35 or a CDR1 of VH;

CDR2-VH comprising SEQ ID NO: 1. 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35 or a CDR2 of VH; and the combination of (a) and (b),

CDR3-VH comprising SEQ ID NO: 1. 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35 or a CDR3 of VH; and/or the presence of a gas in the gas,

(2) the light chain CDRs comprise:

CDR1-VL comprising SEQ ID NO: 2. 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 or a CDR1 of VL;

CDR2-VL comprising SEQ ID NO: 2. 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 or a CDR2 of VL; and the combination of (a) and (b),

CDR3-VL comprising SEQ ID NO: 2. 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, or a CDR3 of a VL as set forth in any one of claims 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36.
The antibody or antigen-binding fragment of claim 1,

(1) the heavy chain variable region and the light chain variable region have sequences shown as SEQ ID NO 1 and SEQ ID NO 2 respectively;

(2) the heavy chain variable region and the light chain variable region have sequences shown in SEQ ID NO 3 and SEQ ID NO 4 respectively;

(3) the heavy chain variable region and the light chain variable region have sequences shown in SEQ ID NO 5 and SEQ ID NO 6 respectively;

(4) the heavy chain variable region and the light chain variable region have sequences shown as SEQ ID NO 7 and SEQ ID NO 8 respectively;

(5) the heavy chain variable region and the light chain variable region have sequences shown in SEQ ID NO 9 and SEQ ID NO 10 respectively;

(6) the heavy chain variable region and the light chain variable region have sequences shown as SEQ ID NO. 11 and SEQ ID NO. 12 respectively;

(7) the heavy chain variable region and the light chain variable region have sequences shown in SEQ ID NO 13 and SEQ ID NO 14, respectively;

(8) the heavy chain variable region and the light chain variable region have sequences shown as SEQ ID NO 15 and SEQ ID NO 16 respectively;

(9) the heavy chain variable region and the light chain variable region have sequences shown in SEQ ID NO 17 and SEQ ID NO 18 respectively;

(10) the heavy chain variable region and the light chain variable region have sequences shown in SEQ ID NO 19 and SEQ ID NO 20, respectively;

(11) the heavy chain variable region and the light chain variable region have sequences shown as SEQ ID NO 21 and SEQ ID NO 22 respectively;

(12) the heavy chain variable region and the light chain variable region have the sequences shown in SEQ ID NO. 23 and SEQ ID NO. 24, respectively;

(13) the heavy chain variable region and the light chain variable region have the sequences shown in SEQ ID NO. 25 and SEQ ID NO. 26, respectively;

(14) the heavy chain variable region and the light chain variable region have the sequences shown in SEQ ID NO 27 and SEQ ID NO 28, respectively;

(15) the heavy chain variable region and the light chain variable region have the sequences shown in SEQ ID NO 29 and SEQ ID NO 30, respectively;

(16) the heavy chain variable region and the light chain variable region have sequences shown as SEQ ID NO 31 and SEQ ID NO 32 respectively;

(17) the heavy chain variable region and the light chain variable region have the sequences shown in SEQ ID NO 33 and SEQ ID NO 34, respectively;

(18) the heavy chain variable region and the light chain variable region have sequences shown in SEQ ID NO 35 and SEQ ID NO 36, respectively; or the like, or, alternatively,

(19) the heavy chain variable region and the light chain variable region each have a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to the sequence shown in (1) to (18) above.
The antibody or antigen-binding fragment of any one of claims 1 to 6, characterized in that the antibody or antigen-binding fragment is: (1) a chimeric antibody or fragment thereof; (2) a fully human antibody or fragment thereof; or, (3) a humanized antibody or fragment thereof.
The antibody or antigen-binding fragment of any one of claims 1 to 6, characterized in that it binds human CD38 with a dissociation constant (KD) of no more than 5nM and binds cynomolgus monkey CD38 with a dissociation constant (KD) of no more than 25 nM.
The antibody or antigen-binding fragment of any one of claims 1 to 6, wherein the antibody comprises the sequence of a constant region of any one of human or murine antibodies IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, or IgD; preferably comprising the sequence of the constant region of the human or murine antibody IgG1, IgG2, IgG3 or IgG 4.
The antibody or antigen-binding fragment of any one of claims 1 to 6, characterized in that the antigen-binding fragment is selected from one or more of F (ab)2, Fab', Fab, Fv, scFv, diabody, nanobody, and antibody minimal recognition unit.
An antibody or antigen-binding fragment that competitively binds to CD38 or an epitope thereof with the antibody or antigen-binding fragment of any one of claims 1-10 and has the following properties:

(1) specifically bind to CD38 recombinant protein and cells expressing CD 38;

(2) mediates antibody-dependent cellular cytotoxicity killing (ADCC) activity;

(3) mediates antibody-dependent cell-mediated phagocytosis (ADCP);

(4) (ii) mediating cross-linking induced cell death;

(5) no or only weak antibody-mediated complement-dependent cytotoxicity (CDC) activity;

(6) inhibiting NAD hydrolase activity; or/and

(7) inhibiting tumor growth.
An isolated nucleic acid molecule encoding the antibody, antigen-binding fragment, or any combination thereof of any one of claims 1-11.
An expression vector comprising the isolated nucleic acid molecule of claim 12.
An isolated host cell comprising the isolated nucleic acid molecule of claim 12, or the expression vector of claim 13; preferably, the host cell is a eukaryotic cell or a prokaryotic cell; more preferably, the host cell is derived from a mammalian cell, a yeast cell, an insect cell, E.coli and/or Bacillus subtilis; more preferably, the host cell is selected from Chinese hamster ovary Cells (CHO).
A method for producing an antibody or antigen-binding fragment, comprising culturing the host cell of claim 14 under suitable conditions and isolating the antibody or antigen-binding fragment.
A pharmaceutical composition comprising the antibody or antigen-binding fragment of any one of claims 1-11, the isolated nucleic acid molecule of claim 12, the expression vector of claim 13, the cell of claim 14, or the product of the process of claim 15, and a pharmaceutically acceptable carrier; preferably, the pharmaceutical composition further comprises an additional antineoplastic agent.
A kit comprising the antibody or antigen-binding fragment of any one of claims 1-11, the isolated nucleic acid molecule of claim 12, the expression vector of claim 13, the cell of claim 14, or the product made by the method of claim 15, and instructions for use.
Use of the antibody or antigen-binding fragment of any one of claims 1 to 11, the isolated nucleic acid molecule of claim 12, the expression vector of claim 13, the cell of claim 14, the product made by the process of claim 15, or the pharmaceutical composition of claim 16 for the manufacture of a medicament for the prevention and/or treatment of a disease associated with aberrant expression of CD38, preferably a tumor.
A method of preventing and/or treating a disease associated with aberrant expression of CD38, comprising administering to a patient in need thereof the antibody or antigen binding fragment of any one of claims 1-11, the isolated nucleic acid molecule of claim 12, the expression vector of claim 13, the cell of claim 14, the product made by the method of claim 15, or the pharmaceutical composition of claim 16; the disease is preferably a tumor.
A Chimeric Antigen Receptor (CAR) comprising at least an extracellular antigen-binding domain comprising the antibody or antigen-binding fragment of any one of claims 1-11, a transmembrane domain, and an intracellular signaling domain.
An immune effector cell expressing the chimeric antigen receptor of claim 20 or comprising a nucleic acid fragment encoding the chimeric antigen receptor of claim 20; preferably, the immune effector cell is selected from a T cell, preferably from a cytotoxic, regulatory or helper T cell, a NK cell, a NKT cell, a DNT cell, a double negative T cell, a monocyte, a macrophage, a dendritic cell or a mast cell; preferably, the immune effector cells are autoimmune effector cells or allogeneic immune effector cells.
A multispecific antibody comprising an antibody or antigen-binding fragment according to any one of claims 1 to 11; preferably, the multispecific antibody further comprises an antibody or antigen-binding fragment that specifically binds to an antigen other than CD38 or binds to a different epitope of CD38 than the antibody or antigen-binding fragment of any one of claims 1-11; preferably, the antigen other than CD38 may be selected from: CD3, preferably CD3 epsilon; CD16, preferably CD 16A; CD 32B; PD-1; PD-2; PD-L1; VEGF; NKG 2D; CD 19; CD 20; CD 40; CD 47; 4-1 BB; CD 137; an EGFR; EGFRvIII; TNF-alpha; CD 33; MSLN; HER 2; HER 3; HAS; CD 5; CD 27; EphA 2; EpCAM; MUC 1; MUC 16; CEA; claudin18.2; a folate receptor; claudin 6; WT 1; NY-ESO-1; MAGE 3; ASGPR1 or CDH 16; more preferably, the multispecific antibody may be bispecific, trispecific or tetraspecific, and the multispecific antibody may be bivalent, tetravalent or hexavalent.
The antibody or antigen-binding fragment of any one of claims 1 to 11, characterized in that the antibody or antigen-binding fragment is further conjugated to a therapeutic agent or tracer; preferably, the therapeutic agent is selected from the group consisting of a radioisotope, a cytotoxic agent or an immunomodulatory agent, and the tracer is selected from the group consisting of a radiocontrast agent, a paramagnetic ion, a metal, a fluorescent label, a chemiluminescent label, an ultrasound contrast agent and a photosensitizer; more preferably, the cytotoxic agent is selected from alkaloids (alkaloids), methotrexate (methotrexate), anthracyclines (doxorubicin) or taxanes (taxanes); the toxin compound is preferably DM1, DM4, SN-38, MMAE, MMAF, Duocarmycin, Calichemicin, DX 8951.
The antibody or antigen-binding fragment of any one of claims 1 to 11, characterized in that the antibody or antigen-binding fragment is further linked to another functional molecule which may be selected from one or more of the following: a signal peptide, protein tag, or cytokine; preferably, the cytokine may be selected from IL-2, IL-6, IL-12, IL-15, IL-21, IFN or TNF-alpha.