CN114616245B - anti-CD 38 antibody and application thereof - Google Patents

Abstract

The invention discloses an antibody or 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 office, application number 201911286272.1, entitled "an anti-CD 38 antibody and use thereof," filed on day 13, 12, 2019, 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 their use for treating diseases associated with abnormal CD38 expression, such as tumors.

Background

CD38 biological Activity

CD38 is a type II transmembrane glycoprotein, and binding of CD38 to its ligand CD31 affects cell migration and hyaluronic acid-interacting receptor-mediated adhesion. The increased expression intensity of CD38 after lymphocyte activation, whose expression is mainly concentrated in hematopoietic cells; is widely present in lymphocytes and bone marrow cells, but is rarely expressed in most mature resting lymphocytes.

CD38 has a variety of biological effects, belongs to ribose cyclase, and can use nicotinamide adenine dinucleotide NAD+ as a substrate to generate ADP ribose and cyclic ADP ribose. This pair of extracellular metabolism, intracellular Ca ²⁺ Has important regulation and control functions such as cell adhesion, signal transduction and the like. The natural ligand for CD38 is CD31/PECAM, which induces tyrosine phosphorylation and downstream signaling upon binding of CD38 and CD31 to regulate lymphocyte proliferation and cytokine release.

In normal humans, the level of CD38 expression is relatively low in bone marrow and lymphocytes and some non-hematopoietic tissue cells, in contrast to high levels of CD38 expression in normal plasma cells and Multiple Myeloma (MM) cells, which makes CD38 a good target for therapeutic antibodies 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 in CD 38-expressing lymphocytes. Simultaneously, CD38 also has extracellular enzyme activity, participates in the generation of nucleotide metabolites, and regulates the storage of intracellular calcium.

CD38 monoclonal antibodies for the treatment of Multiple Myeloma (MM)

anti-CD 38 monoclonal antibodies are targeted antibody drugs against CD38 protein molecules that are 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 apoptosis of tumor cells by cross-linking (programmedcell death). Based on its good tolerability and proven exact efficacy, dratuumab was FDA approved in 2015 for treatment of relapsed/refractory MM patients and was marketed in china in 2019, 7. The second CD38 mab, isatuximab of Sanofi, has made breakthrough progress in clinical phase 3 and pomalidomide in combination with dexamethasone in relapsed/refractory MM patients, and is currently marketed by FDA and EMA applications. Because the novel tumor cell inhibitor has the function of inhibiting NDA hydrolase activity, the novel tumor cell inhibitor can help antagonize the inhibition effect of inhibiting cells and inhibiting factors on T cells in the microenvironment of solid tumor tumors, clinical tests of solid tumors such as liver cancer, head and neck cancer, ovarian cancer, glioblastoma and the like are also carried out in the United states. Morphosys, inc. licensed to the Chinese world company for CD38 mab, decreased from 4-6 hours to about 2 hours, possibly associated with its reduced CDC activity, compared to Daatumumab and Isatuximab, upon clinical intravenous infusion. Currently, MM clinical phase 3 trials are performed in china. We have aimed to develop a class of CD38 antibodies which have equivalent or higher binding affinity for CD38 protein and active functions such as ADCC, ADCP, etc., as compared to Daratumumab and Isatuximab, while having only weak or no CDC activity, but which inhibit NDA hydrolase activity.

Relationship between drug resistance of immune checkpoint inhibitor such as CD38 and PD-L1 mab

Clinical data analysis shows that the drug resistance of immune checkpoint inhibition drugs such as CD38 and PD-L1 antibodies is closely related, and the response rate of patients with high expression of CD38 to PD-L1 antibodies is strongly related. The related mechanism is as follows: 1) adenosinergic pathway is one of the important mechanisms of drug resistance such as PD-L1 antibody. The PD-L1 antibody can cause high expression of CD38 in tumor microenvironment by tumor cells and tregs, and the CD38 promotes the formation of adenosine by catalyzing NAD+, so that T, NK and macrophages are inhibited. The enzyme activity inhibition effect of the CD38 antibody can inhibit the formation of adenosine, and the drug resistance of the PD-L1 antibody can be resisted. 2) CD38 expression on immune suppressor cells such as PD-L1 monoclonal antibody resistant patients, tumor cells, tregs, MDSCs and the like is obviously up-regulated, CD38 is an important mark of the immune checkpoint inhibitor drug resistance such as PD-L1 antibodies and the like, and the immune suppressor cells in tumor microenvironment can be reduced through the killing effect of the CD38 monoclonal antibody, so that the tumor killing effect of immune cells is promoted. Of course, at the same time, consideration is also given to how to design drugs to avoid the killing effect of CD38 antibodies on activated T cells. 3) In addition, the NAD+ hydrolase inhibiting the CD38 protein can promote the differentiation of Th0 cells into anti-tumor T cells, inhibit the conversion of Th0 cells into Treg cells and increase the anti-tumor effect.

Therefore, the anti-tumor monoclonal antibody has tumor killing and NAD hydrolase activity inhibiting effects, can specifically kill CD38 monoclonal antibodies of inhibiting cells such as Treg, MDSC and the like in blood tumor cells and solid tumors which highly express CD38, or on the basis, the anti-CD 38X bispecific antibody or the double-target drug containing the antigen binding fragment of the CD38 monoclonal antibody is developed and used for improving the drug effect of treating MM, or is used for treating other refractory malignant tumors such as solid tumors, in particular solid tumors, and has great social and economic significance.

Disclosure of Invention

The present invention provides antibodies or antigen binding fragments that specifically bind to human CD38, nucleic acids encoding these antibodies, and compositions and uses thereof for killing tumor cells, including the antibodies and antigen binding fragments and pharmaceutical compositions. The antibodies or antigen binding fragments of the invention can bind not only human CD38, but also cynomolgus monkey CD38.

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

(1) The heavy chain CDRs comprise: CDR1-VH, CDR2-VH and CDR3-VH; the CDR1-VH, CDR2-VH and CDR3-VH have any sequence combination selected from the group consisting of:

And;

(2) The light chain CDRs comprise: CDR1-VL, CDR2-VL and CDR3-VL; the CDR1-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 said sequence combination:

each of CDR1-VH, CDR2-VH, CDR3-VH, CDR1-VL, CDR2-VL and CDR3-VL is encoded according to the current analytical methods of KABAT, chothia or IMGT.

In some embodiments, an isolated antibody or antigen binding fragment that specifically binds human CD38 comprises 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, a combination of CDRs having 1, 2, 3, or more amino acid insertions, deletions, and/or substitutions compared to the sequence of the heavy chain CDRs combination.

In some embodiments, an isolated antibody or antigen binding fragment that specifically binds human CD38 comprises a light chain CDRs combination 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, a combination of CDRs having 1, 2, 3 or more amino acid insertions, deletions and/or substitutions compared to the sequence of the combination of light chain CDRs.

In some embodiments, an isolated antibody or antigen-binding fragment that specifically binds human CD38 comprises a combination of heavy and light chain CDRs selected from the group consisting of: VH1+ VL1, VH2+ VL2, VH3+ VL3, VH4+ VL4, VH5+ VL5, VH6+ VL6, VH7+ VL7, VH8+ VL8, VH9+ VL9, VH10+ VL10, VH11+ VL11, VH12+ VL12, VH13+ VL13, VH14+ VL14, VH15+ VL15, VH16+ VL16, VH17+ VL17, VH18+ VL18, VH19+ VL19, VH20+ VL20, VH21+ VL21, VH22+ VL22, VH23+ VL23, VH24+ VL24, VH25+ VL25, VH26+ VL26, VH27+ VL27, VH28+ VL28, VH29+ VL29, VH30+ VL30, VH31+ 31, VH32+ VL32, VH33+ VL33, VH34+ VL34, VH35+ VL35 or VH36+ VL36, and the heavy chain and light chain CDRs have a combination of amino acid, amino acid deletion, or more than the heavy chain and light chain CDRs.

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

CDR2-VH comprising SEQ ID NO: 1. 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35; and, a step of, in the first embodiment,

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

(2) The light chain CDRs comprise:

CDR1-VL comprising SEQ ID NO: 2. CDR1 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;

CDR2-VL comprising SEQ ID NO: 2. 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36; and, a step of, in the first embodiment,

CDR3-VL comprising SEQ ID NO: 2. 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 the amino acid sequence of SEQ ID NO:1 and SEQ ID NO: 2;

(2) The heavy chain variable region and the light chain variable region have the amino acid sequence of SEQ ID NO:3 and SEQ ID NO: 4;

(3) The heavy chain variable region and the light chain variable region have the amino acid sequence of SEQ ID NO:5 and SEQ ID NO: 6;

(4) The heavy chain variable region and the light chain variable region have the amino acid sequence of SEQ ID NO:7 and SEQ ID NO: 8;

(5) The heavy chain variable region and the light chain variable region have the amino acid sequence of SEQ ID NO:9 and SEQ ID NO:10, a sequence shown in seq id no;

(6) The heavy chain variable region and the light chain variable region have the amino acid sequence of SEQ ID NO:11 and SEQ ID NO: 12;

(7) The heavy chain variable region and the light chain variable region have the amino acid sequence of SEQ ID NO:13 and SEQ ID NO:14, a sequence shown in seq id no;

(8) The heavy chain variable region and the light chain variable region have the amino acid sequence of SEQ ID NO:15 and SEQ ID NO:16, a sequence shown in seq id no;

(9) The heavy chain variable region and the light chain variable region have the amino acid sequence of SEQ ID NO:17 and SEQ ID NO:18, a sequence shown in seq id no;

(10) The heavy chain variable region and the light chain variable region have the amino acid sequence of SEQ ID NO:19 and SEQ ID NO:20, a sequence shown in seq id no;

(11) The heavy chain variable region and the light chain variable region have the amino acid sequence of SEQ ID NO:21 and SEQ ID NO: 22;

(12) The heavy chain variable region and the light chain variable region have the amino acid sequence of SEQ ID NO:23 and SEQ ID NO:24, a sequence shown in seq id no;

(13) The heavy chain variable region and the light chain variable region have the amino acid sequence of SEQ ID NO:25 and SEQ ID NO: 26;

(14) The heavy chain variable region and the light chain variable region have the amino acid sequence of SEQ ID NO:27 and SEQ ID NO:28, a sequence shown in seq id no;

(15) The heavy chain variable region and the light chain variable region have the amino acid sequence of SEQ ID NO:29 and SEQ ID NO:30, a sequence shown in seq id no;

(16) The heavy chain variable region and the light chain variable region have the amino acid sequence of SEQ ID NO:31 and SEQ ID NO: 32;

(17) The heavy chain variable region and the light chain variable region have the amino acid sequence of SEQ ID NO:33 and SEQ ID NO: 34;

(18) The heavy chain variable region and the light chain variable region have the amino acid sequence of SEQ ID NO:35 and SEQ ID NO:36, a sequence shown in seq id no;

or alternatively, the first and second heat exchangers may be,

(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 with the sequences shown in (1) to (18) above.

In a preferred embodiment, the antibodies or antigen binding fragments thereof of the invention are:

(1) A chimeric antibody or fragment thereof;

(2) A fully human antibody or fragment thereof; or alternatively, the first and second heat exchangers may be,

(3) Humanized antibodies or fragments thereof.

In a preferred embodiment, the antibody or antigen binding fragment thereof of the invention has a dissociation constant (KD) for binding to human CD38 of no more than 5nM and a dissociation constant (KD) for binding to cynomolgus monkey CD38 of no more than 25nM.

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 the human or murine antibodies IgG1, igG2, igG3, igG4, igA, igM, igE or IgD; preferably comprising the sequence of the constant region of a human or murine antibody IgG1, igG2, igG3 or IgG 4.

In a preferred embodiment, the antigen binding fragments of the invention are selected from one or more of F (ab) 2, fab', fab, fv, scFv, bispecific antibodies, nanobodies and antibody minimal recognition units.

In a preferred embodiment, the antibody or antigen binding fragment thereof of the invention can competitively bind to CD38 or an epitope thereof with an antibody or antigen binding fragment selected from the group consisting of 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 to cells expressing human CD 38;

2) Mediating Antibody Dependent Cellular Cytotoxicity (ADCC) activity;

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

4) Mediate cross-linking induced cell death;

5) No or only weak antibody-mediated Complement Dependent Cytotoxicity (CDC) activity;

6) Inhibiting NAD hydrolase activity; or/and (or)

7) Inhibit 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 compounds are preferably DM1, DM4, SN-38, MMAE, MMAF, duocarmycin, calicheamicin, 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 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.

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 invention provides an expression vector comprising an isolated nucleic acid molecule of the invention described above.

In some embodiments, the invention provides a host cell comprising an isolated nucleic acid molecule or expression vector of the 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 mammalian cells, yeast cells, insect cells, E.coli and/or B.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, culturing a host cell of the invention as described above under appropriate 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 and an antigen-binding fragment) produced by a method of the invention described above, and a pharmaceutically acceptable carrier.

In a preferred embodiment, the pharmaceutical composition further comprises an additional anti-tumor agent.

In some embodiments, the invention provides a method of preventing and/or treating a disease associated with abnormal CD38 expression 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) prepared 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 as described above, an isolated nucleic acid molecule as described above, an expression vector as described above, a cell as described above, a product (e.g., an antibody and antigen binding fragment) prepared by a method as described above, or a pharmaceutical composition as described above, for 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 and antigen-binding fragment) prepared 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 an antigen other than CD38 or binds a CD38 epitope different from 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 CD16A; CD32B; PD-1; PD-2; PD-L1; VEGF; NKG2D; CD19; CD20; CD40; CD47;4-1BB; CD137; EGFR (epidermal growth factor receptor); EGFRvIII; TNF-alpha; CD33; MSLN; HER2; HER3; HAS; CD5; CD27; ephA2; epCAM; MUC1; MUC16; CEA; claudin18.2; a folate receptor; claudin6; WT1; NY-ESO-1; MAGE3; ASGPR1 or CDH16.

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 any antibody or antigen-binding fragment of the invention, a transmembrane domain, and an intracellular signaling domain.

In another aspect, the 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 T cells, preferably from cytotoxic T cells, regulatory T cells or helper T cells, NK cells (natural killer cell), NKT cells (natural killer T cell), DNT cells (double negative T cell), monocytes, macrophages, dendritic cells or mast cells; preferably, the immune effector cell is an autoimmune effector cell or an alloimmune effector cell.

Terminology and definition:

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

The term "CD38" is a glycoprotein with the action of cyclic ADP-ribose hydrolase 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 an immunoglobulin molecule that specifically binds or is immunoreactive with an antigen of interest, including polyclonal, monoclonal, genetically engineered, and other modified forms of antibodies (including, but not limited to, chimeric antibodies, humanized antibodies, fully human antibodies, 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, rIgG, 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 incomplete antibody fragments (e.g., fab and F (ab') 2 fragments), which lack the Fc fragment of the intact antibody (cleared more rapidly from the animal circulation) and thus 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 (affibody), nanobody, aptamer, or domain antibody. Examples of binding fragments that encompass 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 at a hinge region by a disulfide bond; (iii) an Fd fragment consisting of VH and CHl domains; (iv) Fv fragments consisting of the VL and VH domains of the antibody single arm; (V) a dAb comprising VH and VL domains; (vi) dAb fragments consisting of VH domains (Ward et al Nature 341:544-546, 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 connected 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 a single chain Fv (scFv); see, e.g., bird et al, science 242:423-426, 1988, and Huston et al, proc. Natl. Acad. Sci. USA 85:5879-5883, 1988). 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 whole 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 to at least two different antigens. In the present invention, one of the binding specificities may be detected against an epitope of CD38 and the other may be detected against another epitope of CD38 or any other antigen, e.g. against a cell surface protein, a receptor subunit, a tissue specific antigen, a virus-derived protein, a virus-encoded envelope protein, a bacterial-derived protein or a bacterial surface protein, etc.

The term "chimeric" antibody refers to an antibody having a variable sequence derived from an immunoglobulin of one origin (e.g., rat or mouse) and constant regions derived from an immunoglobulin of 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 4:214-221; gilles et al, 1985J Immunol Methods 125:191-202; the above is incorporated by reference herein.

The term "humanized antibody" refers to an antibody which is re-expressed by modifying a murine monoclonal antibody by gene cloning and DNA recombination techniques, and most of the amino acid sequences of the humanized antibody are replaced by human sequences, so that the affinity and the specificity of the parent murine monoclonal antibody are basically reserved, the heterology of the humanized antibody is reduced, and the humanized antibody is favorable for being applied to human bodies. Humanized antibodies are those in which either the constant region portions of the antibody (i.e., the CH and CL regions) or all of the antibody are encoded by human antibody genes. Humanized antibodies can greatly reduce the immune 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 and heavy chain variable domains. The more conserved portions of the variable domains are called the Framework Regions (FR). As understood in the art, the amino acid positions representing the hypervariable regions of an antibody may vary depending on the context and various definitions known in the art. Some positions within the variable domain may be considered heterozygous hypervariable positions, as these positions may be considered to be within a hypervariable region under one set of criteria (e.g. IMGT or KABAT) and outside a hypervariable region under a different set of criteria (e.g. KABAT or IMGT). One or more of these locations may also be found in the extended hypervariable region. The invention includes antibodies comprising modifications in the positions of these heterozygous hypermutations. The variable domains of the natural heavy and light chains each comprise four framework regions, principally in a lamellar configuration, which are linked by three CDRs (CDR 1, CDR2 and CDR 3) that form loops connecting the lamellar structure and in some cases form part of the lamellar structure. The CDRs in each chain are held closely together by the FR regions in sequence FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 and with CDRs from other antibody chains contribute to the formation of the antigen binding site of the antibody (see Kabat et al Sequences of Protein sofImmunological Interest, national Institute of Health, bethesda, md 1987; incorporated herein by reference). For example, herein, CDR1-VH, CDR2-VH and CDR3-VH refer to the first CDR, the second CDR and the third CDR, respectively, of a heavy chain variable region (VH), which three CDRs constitute the CDR combination (VHCDR combination) of the heavy chain (or variable region thereof); CDR1-VL, CDR2-VL and CDR3-VL refer to the first CDR, second CDR and third CDR, respectively, of the light chain variable region (VL) and these three CDRs constitute the CDR combinations (VLCDR combinations) of the light chain (or variable regions thereof).

The term "VH" refers to the variable region of an 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 Fv, scFv, dsFv or Fab).

The term "antibody conjugate" refers to a conjugate body/conjugate formed by the chemical bonding of an antibody molecule to another molecule, either directly or through a linker. Such as an antibody-drug conjugate (ADC), wherein the drug molecule is said another molecule.

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

The term "percent (%) sequence identity" refers to the percentage of amino acid (or nucleotide) residues of a candidate sequence that are identical to amino acid (or nucleotide) residues of a reference sequence after aligning the sequences and introducing gaps, if desired, for maximum percent sequence identity (e.g., gaps may be introduced in one or both of the candidate and reference sequences for optimal alignment, and non-homologous sequences may be ignored for comparison purposes). For the purpose of determining percent sequence identity, the alignment may be accomplished in a variety of ways well known to those skilled in the art, for example using publicly available computer software such as BLAST, ALIGN, or Megalign (DNASTAIi) software. One skilled in the art can determine appropriate parameters for measuring the alignment, including any algorithm that requires maximum alignment over the full length of the sequences being compared. For example, a reference sequence for comparison to a candidate sequence may show that the candidate sequence exhibits from 50% to 100% sequence identity over the entire length of the candidate sequence or over selected portions of consecutive amino acid (or nucleotide) residues of the candidate sequence. The length of the candidate sequences aligned for comparison purposes may be, for example, 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 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 of greater than 100nM (e.g., greater than 500nM, 1 μm, 100 μm, 500 μm, or 1 mM) for the particular antigen or epitope thereof. Antibodies that specifically immunoreact with a particular protein or carbohydrate may be selected using a variety of immunoassay formats. For example, solid phase ELISA immunoassays are routinely used to select antibodies that specifically immunoreact with a protein or carbohydrate. See, harlow & Lane, antibodies, ALaboratory Manual, cold Spring Harbor Press, newYork (1988), and Harlow & Lane, using Antibodies, A Laboratory Manual, cold Spring Harbor Press, newYork (1999), which describe immunoassay formats and conditions that may be used to determine specific immunoreactivity.

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

The term "ADCC" (antibody-dependent cell-mediated cytotoxicity, ADCC), i.e. antibody-dependent cell-mediated cytotoxicity, refers to the specific recognition of an epitope of a tumor cell by the Fab fragment of an antibody, whose Fc fragment binds to an FcR on the surface of a killer cell (NK cell, macrophage, etc.), which mediates direct killing of the target cell by the killer cell. The strength of antibody-mediated ADCC is related to a number of factors, such as the affinity of the antibody for the antigen, the affinity of the antibody for the Fc-segment receptor, the characteristics of immune effector cells, and the like. In general, the stronger the antibody-mediated ADCC that has high affinity for antigen or Fc receptor. Glycosylation and amino acid sequence engineering of the Fc fragment of an antibody can enhance ADCC activity. In glycosylation modification of antibodies, fucose is considered to be the most important sugar affecting ADCC activity, and defucosylation can significantly improve affinity of antibodies to fcγriiia and ADCC activity.

The term "induction of apoptosis by cross-linking of an antibody" (antibody induces programmed cell death via cross-linking) refers to induction of apoptosis processes by the Fc fragment of an antibody with an Fc receptor (FcR) or after the action of a second cross-linked antibody. ADCC and ADCP are the induction of aggregated IgG constant domains (Fc domains) by monoclonal antibody binding to activate fcγrs on immune effector cells such as natural killer cells, macrophages and polymorphonuclear cells. Monoclonal antibodies can enhance the agonistic activity of the antibody or induce apoptosis (programmed cell death, PCD) by the interaction of the Fc segment of the antibody with Fc receptors. Agonist therapeutic monoclonal antibodies targeting members of the death receptor family induce PCD via an exogenous apoptotic pathway. Agonistic monoclonal antibodies that target these death receptors induce PCD enhanced by cross-linking, such as by a second cross-linked antibody or more physiologically binding to fcγrs. Antibody-mediated antigen cross-linking, unrelated to the death receptor family, can also induce PCD, but cannot pass through the classical apoptotic pathway. This pathway is characterized by homotypic aggregation of cells, including cytoskeletal recombination, lysosomal activation, and reactive oxygen species production. Such non-induced PCD pathways may be enhanced by Fc-crosslinked secondary antibodies or fcγr 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). A variety of vectors have been developed for delivering polynucleotides encoding exogenous proteins into prokaryotic or eukaryotic cells. The expression vectors of the invention contain polynucleotide sequences and additional sequence elements, for example, for expressing proteins and/or integrating these polynucleotide sequences into the genome of mammalian cells. Certain 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 transcription of genes. Other useful vectors for expressing antibodies and antibody fragments contain polynucleotide sequences that enhance the translation rate of these genes or improve the stability or nuclear export of mRNA produced by gene transcription. These sequence elements include, for example, 5 'and 3' untranslated regions, internal Ribosome Entry Sites (IRES) and polyadenylation signal sites, in order to direct efficient transcription of genes carried on expression vectors. The expression vectors of the invention may also contain polynucleotides encoding markers for selecting cells containing such vectors. Examples of suitable markers include genes encoding antibiotic (e.g., ampicillin, chloramphenicol, kanamycin, or nociceptin) resistance.

The terms "subject," "subject," and "patient" refer to an organism that is receiving treatment for a particular disease or disorder (e.g., cancer or infectious disease) as described herein. Examples of subjects and patients include mammals such as humans, primates, pigs, goats, rabbits, hamsters, cats, dogs, guinea pigs, members of the bovine family (e.g., cattle, bison, buffalo, elk, and yaks, etc.), cows, sheep, horses, and bisons, 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 a surgical or pharmaceutical treatment (surgical or therapeutic treatment) that is intended to prevent, slow down (reduce) the progression of an undesired physiological change or disorder, such as a cell proliferative disorder (e.g., cancer or infectious disease), in a subject. 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 with the condition or disease and subjects prone to the condition or disease or subjects intended to prevent the condition or disease. When referring to terms slow down, alleviate, attenuate, mitigate, alleviate, etc., the meaning also includes eliminating, vanishing, non-occurrence, etc.

The term "effective amount" refers to an amount of a therapeutic agent that is effective to prevent or ameliorate a disease condition or 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, such as treating, curing, preventing or alleviating a related medical condition, or an increase in the rate of treating, curing, preventing or alleviating such conditions. When an active ingredient is administered to an individual alone, a therapeutically effective dose is referred to as the ingredient alone. When a combination is used, a therapeutically effective dose refers to the combined amounts of the active ingredients that produce a therapeutic effect, whether administered in combination, sequentially or simultaneously.

The term "cancer" refers to or describes a physiological condition in a mammal that is typically characterized by unregulated cell growth. Included in this definition are benign and malignant cancers. As used herein, the term "tumor" or "tumor" refers to all neoplastic (neoplastic) cell growth and proliferation, whether malignant or benign, and all pre-cancerous 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 purposes of illustrating some preferred embodiments of the invention, however, it is to be understood that the invention is not limited to the specific embodiments disclosed.

FIG. 1A, CD-His binding titer assay with human CD38-His in serum of His immunized mice;

FIG. 1B, CD-His-immunized mouse serum and human EGFR-His binding titer assay;

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

FIG. 2B, CD-His-immunized mice spleen B cells CREG-His staining results;

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

FIG. 3A, FACS detection of direct binding EC50 values for anti-CD 38 candidate antibodies and Daudi cells;

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

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

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

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

FIG. 6A, dose-dependent relationship of anti-CD 38 antibodies to Annexin V positive cells induced by cross-linking, ratio higher than PI positive cell ratio;

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

FIGS. 7A-7B, anti-CD 38 antibody mediated ADCC activity assays of NK cells against Daudi cells;

FIGS. 8A-8C, anti-CD 38 antibody mediated ADCC activity assays of PBMC on Daudi cells;

FIG. 9, detection of anti-CD 38 antibodies inhibiting NAD hydrolase activity of CD38 protein;

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

Detailed Description

The present invention will now be described in detail with reference to the examples and the accompanying drawings, which are provided herein for the purpose of illustrating some preferred embodiments of the invention, however, it is to be understood that the invention is not limited to the specific embodiments disclosed or to the extent that it is not limited thereto. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

EXAMPLE 1 mouse immunization to generate monoclonal antibodies that specifically bind CD38

A first immunization was performed on 6-8 week old female SJL mice (purchased from Beijing Vitrendylar laboratory animal technologies Co., ltd.) or Balb/c mice (purchased from Shanghai Laek laboratory animal Co., ltd.) using human CD38-His (Novoprotein, cat: CU 65) with Freund's complete adjuvant (Sigma, cat: F5881); post-three immunizations were performed using the above-described human CD38-His with Freund's incomplete adjuvant (incomplete freund's adjuvant, IFA, sigma, cat: F5506) plus unmethylated cytosine-guanine dinucleotide (CpGODN 1826, synthesized from Shanghai Biotechnology); the immunization was injected with 50. Mu.g/dose of a homogeneous stable emulsion formed by the emulsification procedure. In particular, the first and second immunization followed by the foot pad and back, and the third and fourth immunization followed by the tail subcutaneous and back, to obtain high titer high affinity high specificity antisera and specific immune cells. On days 5-7 after the last immunization (fourth immunization), mice were euthanized and spleens were removed aseptically, mice spleen lymphocytes were isolated aseptically, and were aliquoted into cryopreservation tubes, frozen in liquid nitrogen. The mice were subjected to blood sampling procedures at 10 days after the second immunization and 10 days after the third immunization and the day of euthanasia, serum was separated, and the titer of the anti-CD 38 specific antibody in the serum was measured by using an enzyme-linked immunosorbent assay (ELISA) method.

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

EXAMPLE 2 flow cytometric fluorescence sorting (FACS) of CD38 specific single B cells

Mouse spleen cells immunized with CD38 protein are indirectly labeled with antibody anti-His-APC (R) via antigen CD38-His protein (CD 38-His, novoprotein, cat: CU 65)&D Systems, cat.ic 050a) and antibodies against mouse B cell-specific markers (anti-mouse B220-pacfacial Blue, BD Biosciences, cat.558108; anti-mouse IgD-PE, BD Biosciences, cat.558597; anti-mouse IgM-PE cy7, BD Biosciences, cat.552867) staining and addition of dye 7-AAD (BD Biosciences, cat.51-68981E) to differentiate between dead and living cells prior to sorting, CD38 specific single B cells (7 AAD) were sorted with an AriaIII (BD Co.) flow cell sorter ^- B220 ⁺ IgD ^- IgM ^- CD38 ⁺ ) Into PCR wells containing cell lysate, rnase inhibitor, one cell per well was collected. The results showed that no CD38 was detected in spleens of non-immunized control mice immunized with CD38-His protein (FIG. 2A) or CD38 protein stained with the unrelated protein CREG-His (FIG. 2B) ⁺ B cells, whereas the spleen of CD38-His immunized mice (FIG. 2C) detected a distinct population of CD38 ⁺ B cells, every 10 ⁶ About 65 CD38 in each spleen cell ⁺ B cells.

EXAMPLE 3 amplification and high throughput expression of monoclonal antibodies

The patent 'a combined primer for nested amplification and application thereof' patent application number: 201811618134.4 mRNA from a single cell is reverse transcribed into cDNA as described in example 1. Then, nested PCR was performed using cDNA as a template, and heavy chain and light chain amplification of antibodies was performed, respectively. Amplifying to obtain a heavy chain variable region and a light chain variable region of the antibody, and cloning the heavy chain variable region and the light chain variable region into 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 expression vector and the light chain expression vector are derived from human IgG1. 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-Vκ -Cκ. The single B cell antibody cloning and expression described above all achieved rapid identification and discovery of antibodies in a high throughput manner in 96-well plates. After a series of physicochemical and functional screening 324, 18 positive candidate antibody molecules were obtained in total from the cloning of the expressed heavy and light chains of the antibodies, the CDRs of which were analysed by IMGT and KABAT software respectively, and the corresponding sequence information is shown in table 1 below, wherein table 1 shows the VH and VL sequences of the candidate antibody molecules and table 2 shows the IMGT and KABAT analysis results of the candidate antibody molecules.

The candidate anti-CD 38 antibodies were sequenced and specific sequence information for the heavy and light chain variable regions were as follows:

TABLE 1 specific sequence information for the heavy and light chain variable regions of anti-CD 38 antibodies

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

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

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Example 4 Octet detection of antibody binding affinity and specificity

Using an Octet HTX detection system, the antibody was diluted to 1. Mu.g/ml with PBST, the antigen CD38-His (Novoprotein, cat: CU 65) was diluted to 100nM with PBST as the starting concentration, 2-fold gradient dilution, 7 gradients total, and 96-well Octet plates (Greiner, cat. 655209), 300. Mu.l/well were added sequentially; a cycling program was set up for a total of 8 protein A probes, each cycle loaded with antibody to a probe to a height of 1nm for about 3 minutes, then the probes bound the gradient diluted CD38-His antigen for about 10 minutes to a saturation plateau, and finally regenerated with Glycine pH 1.5. The experimental data were analyzed to fit the equilibrium dissociation constant KD of the antibody antigen and determine the binding rate constant ka and dissociation rate constant KD.

The affinities of the antibody candidates for human and cynomolgus CD38 proteins are summarized in table 3. Binding rate Kon, dissociation rate Kdis, dissociation constant KD. It can be seen from table 3 that the candidate CD38 antibodies have a significantly improved binding affinity to humans compared to the commercially available drug control antibody Daratumumab (Janssen, cat GIS 0503), but a weaker binding affinity to cynomolgus monkeys than Daratumumab, and a substantially equivalent or improved binding affinity to human and cynomolgus monkeys compared to the CD38 mab positive control Mor202-kaps (Biointron, cat.b 4163) prepared according to the published sequences of the morphos patent.

TABLE 3 Octet detection of CD38 antibody binding affinity and specificity

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

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

2 cell lines with high expression of CD38 are selected in the experiment: daudi (purchased from China academy of sciences cell bank, cat: TCHu 140) and CHO-38 cells (ordered from Yongshan Biotechnology Co., ltd.) which express the CD38 protein on the surface of CHO cells. In a U-shaped round bottom 96-well plate, 1×10 ⁵ Daudi or CHO-38 cells and serial dilutions of anti-CD 38 antibody, starting at 10. Mu.g/ml, 10-fold gradient, 6 gradients per antibody, after half an hour incubation at 4℃and 5. Mu.g/ml of secondary anti-hIgG Fc (Invitrogen, cat: 31125) were added and after half an hour incubation at 4℃were run on machine FACS detection.

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

TABLE 4 EC50 results graphs

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 antibodies

ADCP was measured using Daudi cells (available from the national academy of sciences cell Bank, catalog number: TCHu 140) as target cells and human macrophages (induced by PBMC-isolated monocytes) as effector cells.

Preparation of effector cells: frozen normal human peripheral blood mononuclear cells PBMC (Stemexpress, cat: PB 0004C) were thawed and incubated overnight in RPMI1640 (Gibco, cat: 22400-089) supplemented with 100IU/ml rhIL-2 (Peprotech, cat: -200-02). Thereafter, living cell counts were performed after the cells were collected. Monocytes are then isolated from PBMC, induced to differentiate in RPMI1640 containing 10% FBS (Gibco, cat: 10099-141) supplemented with 100 μg/ml GM-CSF (Peprotech, cat:300-03-20 Ug), in 5% CO, with specific reference to the monocyte isolation kit instructions (easy Sep Cat: 19058) ₂ After one week of culture in 37℃culture, the cells were counted after resuspension and used as effector cells.

Preparation of target cells: target cells Daudi (available from the national academy of sciences cell Bank, catalog number: TCHu 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 a U-shaped round bottom 96-well plate, 5×10 ⁴ Daudi cells and dilutedanti-CD 38 antibody at a final concentration of 1. Mu.g/ml, 0.1. Mu.g/ml, pre-incubated at 4℃for half an hour, then added 2X 10 ⁵ Effector cells (effector cells: target cells=4:1), incubated at 4 ℃ for 3 hours. After the incubation is completed, a staining operation is performed.

Dyeing: human TruStain FcX (Biolegend, cat: 422302) was blocked for half an hour, stained antibody APC Mouse anti human CD19 (BD, cat: 555415), AF488mouseanti human CD11b (BD, cat: 557701) was added and incubated at 4℃for half an hour. DPBS (Hyclone, cat: SH 30256.01) containing 2% FBS (Gibco, cat: 10099-141) was washed twice and resuspended, and PI (Invitrogen, cat: P3566) was added before loading and analyzed by flow cytometry.

Cell lysis rate due to ADCP activity was calculated according to the following calculation formula:

phagocytosis rate = ratio of phagocytosed cells (CD 19 ⁺ CD11b ^- ) Percentage of target cells (Celltrace) ⁺ )*100％

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

EXAMPLE 7 analysis of programmed cell death induced by crosslinking of anti-CD 38 antibodies

CD38 antibody cross-linking induced apoptosis was measured on Romas cells (Shanghai Biotechnology Co., ltd., cat. No. CC-Y1430). In a U-shaped round bottom 96-well plate, 4×10 ⁴ Romas 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 pre-incubated for half an hour, then 5. Mu.g/ml anti-hIgG Fc (Invitrogen, cat: 31125) was added at 5% CO ₂ The cells were cultured at 37℃for 20 hours. Cells were stained with Annexin V and PI in a programmed cell death detection kit (eBioscience, cat: BMS500 FI/300), respectively, and analyzed by flow cytometry. The Annexin V positive percentage and PI positive percentage were calculated and analyzed using Flowjo software.

The results show that all candidate CD38 antibodies (0.1 mug/ml) can induce the Romas cells to carry out phosphatidylserine translocation under the action of Fc crosslinking secondary antibody anti hIgGFc (FIG. 5A), so that the positive proportion of Annexin V of the Romas cells is obviously improved. The positive proportion of PI cells was also significantly increased, i.e. death of the cells occurred (fig. 5B). The proportion of Annexin V positive cells was higher than that of PI positive cells, and the effect of different concentrations of CD38 antibodies on cells was further observed (fig. 6A-6B), and it was found that the candidate anti-CD 38 antibodies of the invention were effective in inducing apoptosis in the presence of Fc crosslinking reagents at concentrations of 0.01-10 μg/ml.

Example 8 ADCC Activity test of anti-CD 38 antibodies

Preparation of effector cells: frozen normal human peripheral blood mononuclear cells PBMC (Stemexpress, cat: PB 0004C) were thawed and incubated overnight in RPMI1640 (Gibco, cat: 22400-089) supplemented with 100IU/ml rhIL-2 (Peprotech, cat: -200-02). Thereafter, living cell counts were performed after the cells were collected. NK cells were then isolated and purified from PBMC, specifically NK cells were isolated (Stemcell Cat: 17955) with reference to NK cell isolation kit instructions, and counted as effector cells after the NK cells were resuspended in RPMI1640 supplemented with 10% FBS (Gibco, cat: 10099-141). PBMCs can be used directly for effector cells of ADCC experiments.

Preparation of target cells: target cells Daudi (available from the national academy of sciences cell Bank, catalog number: TCHu 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.

anti-CD 38 antibodies were diluted with RPMI1640 supplemented with 10% FBS, diluted antibodies were plated in 50. Mu.l/Kong Fendao U-bottom 96-well plates, and labeled target cells were then added to the wells. The board is heated to 37 ℃ and 5 percent CO ₂ The incubator was pre-incubated for half an hour. Subsequently, effector cells were added to the wells (effective target ratio NK: daudi 2:1-4:1; PBMC: daudi 20:1-40:1), 37℃and 5% CO ₂ Incubate in incubator for 3-4 hours. Plates were removed, and after adding dead cell marker dye PI (1 μl/well) to each well, flow analysis was performed to measure the proportion of dead cells, i.e., PI positivity, in CelltraceTMviolet positive target cells. Calculation of the ADCC Activity-induced ADCC Activity according to the following calculation formulaIs not limited by the cell lysis rate:

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

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

Example 9 detection of inhibition of CD38 proteolytic enzyme activity by CD38 antibodies

This example uses a CD38 hydrolase activity inhibitor screening kit (BPS biconscience, cat. 79287) to perform the procedure described herein. After adding a hydrolysis buffer solution, 1 mug/ml of antibody to be detected and 0.5 mug/ml of CD38 protein into a 96-well plate, putting the system into a 37 ℃ incubator for incubation for 30 minutes, then adding epsilon-NAD substrate, performing fluorescence detection by an enzyme-labeled instrument, wherein the excitation wavelength is 300nm, the detection wavelength is 410nm, and reading the fluorescence value of each sample hole on the plate every 3 minutes for 60 minutes. Selecting a previous time point for entering a platform phase according to the time-fluorescence value curve, and calculating the inhibition rate:

Inhibition ratio (%) = (Sample fluorescence value-Blank fluorescence value)/(Positive Control fluorescence value-Blank fluorescence value) ×100%.

As shown in fig. 9, the response curves of the control Drug Daratumumab (Dara) and the negative control antibody group IgG1 κ and Drug0 were coincident, and did not inhibit CD38 hydrolase activity; the response curve of the control Isatuximab (Isa) with the function of inhibiting the activity of the CD38 hydrolase is similar to that of the anti-CD 38 antibodies 102 and 215, and the reaction rate of the activity of the CD38 hydrolase is obviously reduced, which indicates that the tested anti-CD 38 antibody can effectively inhibit the activity of the CD38 hydrolase.

Example 10 CDC Activity assay of CD38 antibody

The following cells and antibodies were added to 96-well plates: daudi cells 1X 10 ⁵ cell/well, 25 μl/well, CD38 antibody: 25 μl/well, 4 μg/ml initial concentration, 1:10 dilution, incubation at 37℃for 30 min. Human serum (normal human serum for physical examination in the pharmaceutical industry) was added to give a final concentration of 5%. Incubate at 37℃for 2.5 hours. PI dye (Invitrogen, lot: 1887160) was diluted 1:20 with RPMI 1640, 20. Mu.l/well was added to give a final PI dose of 1. Mu.l/well, and after 5 min incubation at room temperature, the flow cytometer was used to detect and record the proportion of dead cells. CDC activity (%) =pi positive cell proportion. As a result, as shown in fig. 10A-10B, daratumumab, which is known to have strong CDC activity, showed dose-dependent CDC activity as expected, whereas the CD38 candidate antibodies tested had no significant CDC activity except for antibodies 10, 31, 38, 102, 215 at the highest three concentrations. These antibodies were shown to be promising for avoiding intravenous infusion-related reactions due to CDC activity (infusion related reaction, IRR).

Claims (33)

1. An isolated antibody or antigen-binding fragment that specifically binds human CD38, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region (VH) and a light chain variable region (VL), the heavy chain variable region, the light chain variable region comprising a combination of CDRs selected from the group consisting of:

(1) Under IMGT numbering, CDR1-VH is shown as SEQ ID NO.37, CDR2-VH is shown as SEQ ID NO.51, CDR3-VH is shown as SEQ ID NO.52, CDR1-VL is shown as SEQ ID NO.100, CDR2-VL is shown as SEQ ID NO.101, and CDR3-VL is shown as SEQ ID NO. 102; or KABAT number, CDR1-VH is shown as SEQ ID NO.73, CDR2-VH is shown as SEQ ID NO.83, CDR3-VH is shown as SEQ ID NO.69, CDR1-VL is shown as SEQ ID NO.119, CDR2-VL is shown as SEQ ID NO.120, and CDR3-VL is shown as SEQ ID NO. 102;

(2) Under IMGT numbering, CDR1-VH is shown as SEQ ID NO.57, CDR2-VH is shown as SEQ ID NO.58, CDR3-VH is shown as SEQ ID NO.59, CDR1-VL is shown as SEQ ID NO.112, CDR2-VL is shown as SEQ ID NO.113, and CDR3-VL is shown as SEQ ID NO. 114; or KABAT number, CDR1-VH is shown as SEQ ID NO.89, CDR2-VH is shown as SEQ ID NO.90, CDR3-VH is shown as SEQ ID NO.91, CDR1-VL is shown as SEQ ID NO.126, CDR2-VL is shown as SEQ ID NO.127, CDR3-VL is shown as SEQ ID NO. 114;

(3) Under IMGT numbering, CDR1-VH is shown as SEQ ID NO.60, CDR2-VH is shown as SEQ ID NO.58, CDR3-VH is shown as SEQ ID NO.61, CDR1-VL is shown as SEQ ID NO.112, CDR2-VL is shown as SEQ ID NO.113, and CDR3-VL is shown as SEQ ID NO. 115; or KABAT number, CDR1-VH is shown as SEQ ID NO.92, CDR2-VH is shown as SEQ ID NO.93, CDR3-VH is shown as SEQ ID NO.94, CDR1-VL is shown as SEQ ID NO.126, CDR2-VL is shown as SEQ ID NO.127, and CDR3-VL is shown as SEQ ID NO. 115.

2. The antibody or antigen-binding fragment of claim 1, wherein the heavy chain variable region and the light chain variable region comprise a combination of CDRs selected from the group consisting of:

(1) CDRs in the sequences shown in SEQ ID NO. 25 and SEQ ID NO. 26;

(2) CDRs in the sequences shown in SEQ ID NO. 27 and SEQ ID NO. 28;

(3) CDRs in the sequences shown in SEQ ID No. 29 and SEQ ID No. 30.

3. The antibody or antigen-binding fragment of claim 2, wherein the heavy chain variable region and the light chain variable region are selected from the group consisting of:

(1) The heavy chain variable region and the light chain variable region are shown in SEQ ID NO. 25 and SEQ ID NO. 26, respectively;

(2) The heavy chain variable region and the light chain variable region are shown in SEQ ID NO. 27 and SEQ ID NO. 28, respectively;

(3) The heavy chain variable region and the light chain variable region are shown in SEQ ID NO. 29 and SEQ ID NO. 30, respectively.

4. The antibody or antigen-binding fragment of any one of claims 1-3, wherein 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.

5. The antibody or antigen-binding fragment of claim 4, which binds to human CD38 with a dissociation constant (KD) of no more than 5nM and to cynomolgus monkey CD38 with a dissociation constant (KD) of no more than 25nM.

6. The antibody or antigen-binding fragment of claim 5, wherein the antibody comprises the sequence of the constant region of any one of human or murine antibodies IgG1, igG2, igG3, igG4, igA, igM, igE, or IgD.

7. The antibody or antigen-binding fragment of claim 6, comprising the sequence of the constant region of human or murine antibodies IgG1, igG2, igG3 or IgG 4.

8. The antibody or antigen-binding fragment of claim 5, wherein the antigen-binding fragment is selected from one or more of F (ab) 2, fab', fab, fv, scFv, bispecific antibodies.

9. An isolated nucleic acid molecule encoding the antibody or antigen-binding fragment of any one of claims 1-8.

10. An expression vector comprising the isolated nucleic acid molecule of claim 9.

11. An isolated host cell comprising the isolated nucleic acid molecule of claim 9, or the expression vector of claim 10.

12. The host cell of claim 11, wherein the host cell is a eukaryotic cell or a prokaryotic cell.

13. The host cell of claim 12, wherein the host cell is derived from a mammalian cell, a yeast cell, an insect cell, escherichia coli, and/or bacillus subtilis.

14. The host cell of claim 13, wherein the host cell is selected from the group consisting of chinese hamster ovary Cells (CHO).

15. A method of producing an antibody or antigen-binding fragment, wherein the host cell of any one of claims 11-14 is cultured under suitable conditions and the antibody or antigen-binding fragment is isolated.

16. A pharmaceutical composition comprising the antibody or antigen-binding fragment of any one of claims 1-8, the isolated nucleic acid molecule of claim 9, the expression vector of claim 10, the cell of any one of claims 11-14, or the product made by the method of claim 15, and a pharmaceutically acceptable carrier.

17. The pharmaceutical composition of claim 16, wherein the pharmaceutical composition further comprises an additional antineoplastic agent.

18. A kit comprising the antibody or antigen-binding fragment of any one of claims 1-8, the isolated nucleic acid molecule of claim 9, the expression vector of claim 10, the cell of any one of claims 11-14, or the product of the method of claim 15, and instructions for use.

19. Use of an antibody or antigen binding fragment according to any one of claims 1 to 8, an isolated nucleic acid molecule according to claim 9, an expression vector according to claim 10, a cell according to any one of claims 11 to 14, a product prepared by a method according to claim 15, or a pharmaceutical composition according to any one of claims 16 to 17 for the preparation of a medicament for the prevention and/or treatment of a disease associated with abnormal expression of CD 38.

20. The use of claim 19, wherein the disease is a tumor.

21. 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 1-8, a transmembrane domain, and an intracellular signaling domain.

22. An immune effector cell expressing the chimeric antigen receptor of claim 21, or comprising a nucleic acid fragment encoding the chimeric antigen receptor of claim 21.

23. The immune effector cell of claim 22, wherein the immune effector cell is selected from the group consisting of a T cell, NK cell (natural killer cell), NKT cell (natural killer T cell), DNT cell (double negative T cell), monocyte, macrophage, dendritic cell, or mast cell.

24. The immune effector cell of claim 23, wherein the T cell is selected from the group consisting of a cytotoxic T cell, a regulatory T cell, and a helper T cell.

25. The immune effector cell of claim 24, wherein the immune effector cell is an autoimmune effector cell or an alloimmune effector cell.

26. A multispecific antibody comprising the antibody or antigen-binding fragment of any one of claims 1-8.

27. The multispecific antibody of claim 26, further comprising an antibody or antigen-binding fragment that specifically binds an antigen other than CD38 or binds a CD38 epitope different from the antibody or antigen-binding fragment of any one of claims 1-8.

28. The multispecific antibody of claim 26 or 27, wherein the multispecific antibody is bispecific, trispecific, or tetraspecific, and the multispecific antibody is bivalent, tetravalent, or hexavalent.

29. The antibody or antigen-binding fragment of any one of claims 1-8, wherein the antibody or antigen-binding fragment is further conjugated to a therapeutic agent or tracer.

30. The antibody or antigen-binding fragment of claim 29, wherein the therapeutic agent is selected from the group consisting of a radioisotope, a cytotoxic agent, and an immunomodulatory agent, and the tracer agent 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.

31. The antibody or antigen-binding fragment of claim 30, wherein the cytotoxic agent is selected from the group consisting of alkaloids (alloids), methotrexate (methotrexite), anthracyclines (doxorubicin) and taxanes (taxanes).

32. The antibody or antigen-binding fragment of any one of claims 1-8, wherein the antibody or antigen-binding fragment is further linked to another functional molecule selected from one or more of the following: signal peptide, protein tag, or cytokine.

33. The antibody or antigen-binding fragment of claim 32, wherein the cytokine is selected from the group consisting of IL-2, IL-6, IL-12, IL-15, IL-21, IFN, and TNF-alpha.