Anti-GPC3 antibody, anti-GPC3 chimeric antigen receptor and GPC3/CD3 bispecific antibody
12492252 ยท 2025-12-09
Assignee
Inventors
- Avanish VARSHNEY (Westford, MA, US)
- Kehao ZHAO (Newton, MA, US)
- Li ZHOU (West Roxbury, MA, US)
- Xin KAI (Woburn, MA, US)
- Liangjun WEI (Nanjing, CN)
- Ninghai Wang (Woburn, MA, US)
Cpc classification
A61K40/4261
HUMAN NECESSITIES
C07K16/28
CHEMISTRY; METALLURGY
A61K2239/38
HUMAN NECESSITIES
A61K40/11
HUMAN NECESSITIES
C12N15/63
CHEMISTRY; METALLURGY
A61P35/00
HUMAN NECESSITIES
International classification
C07K16/28
CHEMISTRY; METALLURGY
A61K40/11
HUMAN NECESSITIES
A61P35/00
HUMAN NECESSITIES
Abstract
Provided herein are novel Glypican 3 (GPC3) antibodies or antigen binding fragments and GPC3/CD3 bispecific antibodies. The present application also provides chimeric antigen receptors comprising the antibodies or antigen-binding fragments, related CAR-T cells, and preparation methods and uses of the same. The present application further provides pharmaceutical compositions comprising GPC3 antibodies or antigen binding fragments, related GPC3/CD3 bispecific antibodies, related GPC3 CAR or CAR-T cells, and methods of treating cancer in a subject in need thereof by administering the Glypican 3 (GPC3) antibodies or antigen binding fragments, the bispecific antibodies, the chimeric antigen receptors, the CAR-T cells, or the pharmaceutical compositions. The cancers treated in accordance with the application include Glypican-3-positive cancers.
Claims
1. An antibody or antigen binding fragment thereof that specifically binds to a GPC3 protein, the antibody or antigen binding fragment thereof comprising a light chain variable region and a heavy chain variable region, wherein the heavy chain variable region comprises CDR1 having an amino acid sequence represented by SEQ ID NO. 1, CDR2 having an amino acid sequence represented by SEQ ID NO. 2 and CDR3 having an amino acid sequence represented by SEQ ID NO. 3, and the light chain variable region comprises CDR1 having an amino acid sequence represented by SEQ ID NO. 4, CDR2 having an amino acid sequence represented by SEQ ID NO. 5 and CDR3 having an amino acid sequence represented by SEQ ID NO. 6.
2. The antibody or antigen binding fragment thereof of claim 1, wherein the heavy chain variable region comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO. 7, and/or the light chain variable region comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO. 8 or SEQ ID NO. 9.
3. A bispecific antibody, comprising a first antibody or antigen binding fragment comprising the antigen binding fragment of claim 1, and a second antigen binding fragment that specifically binds to one or more subunits or structural domains of CD3.
4. The bispecific antibody of claim 3, wherein the first antibody or antigen binding fragment comprises two identical heavy chains and two identical light chain, and the second antigen binding fragment comprises two identical single chain antibody fragments (scFv), and wherein each of the light chains of the first antibody or antigen binding fragment is fused to the single chain antibody fragment (scFv) of the second antigen binding fragment directly or via a linker.
5. A chimeric antigen receptor (CAR), comprising the antibody or the antigen-binding fragment thereof of claim 1.
6. An isolated nucleic acid comprising a nucleic acid sequence encoding the antibody or antigen binding fragment thereof of claim 1.
7. A vector comprising the nucleic acid of claim 6.
8. An isolated host cell comprising the nucleic acid of claim 6.
9. A pharmaceutical composition, comprising the antibody or antigen binding fragment thereof of claim 1.
10. A method of treating cancer in a subject in need thereof, comprising administering to the subject the antibody or antigen binding fragment thereof of claim 1.
11. The antibody or antigen binding fragment of claim 1, wherein the antigen binding fragment is selected from a Fab fragment, a Fab fragment, a Fab-SH fragment, a F(ab)2 fragment, a Fv fragment and a scFv fragment.
12. The bispecific antibody of claim 3, wherein the second antigen binding fragment is selected from a scFv fragment, a Fv fragment, a F(ab).sub.2 fragment and a Fab fragment.
13. The bispecific antibody of claim 4, wherein the single chain antibody fragment (scFv) comprises: a heavy chain variable region comprising an amino acid sequence of SEQ ID NO. 10, and a light chain variable region comprising an amino acid sequence of SEQ ID NO. 11; a heavy chain variable region comprising an amino acid sequence of SEQ ID NO. 12, and a light chain variable region comprising an amino acid sequence of SEQ ID NO. 11; or a heavy chain variable region comprising an amino acid sequence of SEQ ID NO. 14, and a light chain variable region comprising an amino acid sequence of SEQ ID NO. 11.
14. The CAR of claim 5, sequentially comprising the antibody or antigen-binding fragment thereof, an extracellular hinge region, a transmembrane region, and an intracellular signaling region.
15. The CAR of claim 14, wherein the extracellular hinge region is a CD8 hinge region, the transmembrane region is a CD8 transmembrane region, and the intracellular signaling region is 4-1BB and CD32.
16. The CAR of claim 15, wherein the extracellular hinge region comprises a CD8 hinge region as set forth in SEQ ID NO: 30, the transmembrane region comprises a CD8 transmembrane region as set forth in SEQ ID NO: 31, and the intracellular signaling region comprises 4-1BB as set forth in SEQ ID NO: 32 and CD35 as set forth in SEQ ID NO: 33.
17. The pharmaceutical composition of claim 9, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.
18. The method of claim 10, wherein the cancer is a Glypican-3-positive cancer.
19. The method of claim 18, wherein the cancer is a solid cancer.
20. The method of claim 19, wherein the cancer is liver cancer, melanoma, ovarian clear cell carcinoma, yolk sac tumor or neuroblastoma.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
(1) The novel features of the application are set forth with particularity in the appended claims. Some of the features and advantages of the present application are explained in the following detailed description in the embodiments and in the examples.
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SEQUENCE LISTING
(23) This application contains a Sequence Listing that has been submitted electronically as an ASCII text file named 48644-0011US1. txt. The ASCII text file, created on Dec. 19, 2022, is 73,728 bytes in size. The material in the ASCII text file is hereby incorporated by reference in its entirety.
DETAILED DESCRIPTION OF THE INVENTION
(24) Unless defined otherwise, technical and scientific terms used herein have the same meaning as generally used in the art to which this disclosure belongs. For purposes of interpreting this specification, the following definitions will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa. As used herein and in the appended claims, the singular forms a, an, and the also refer to the plural forms unless the context clearly dictates otherwise, e.g., reference to a host cell includes a plurality of such host cells.
(25) As used herein, the term antigen binding fragment or antigen binding molecule refers in its broadest sense to a molecule that specifically binds an antigenic determinant. Examples of antigen binding molecules are antibodies, antibody fragments and scaffold antigen binding proteins. The term antibody herein is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, monospecific and multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity.
(26) The term antibody as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and/or bind the same epitope, except for possible variant antibodies, e.g., containing naturally occurring mutations or arising during production of a monoclonal antibody preparation, such variants generally being present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen.
(27) A humanized antibody is also called a reshaped human antibody. Specifically, humanized antibodies prepared by grafting the CDR of a non-human animal antibody such as a mouse antibody to a human antibody and such are known. Common genetic engineering techniques for obtaining humanized antibodies are also known. Specifically, for example, overlap extension PCR is known as a method for grafting a mouse antibody CDR to a human FR. In overlap extension PCR, a nucleotide sequence encoding a mouse antibody CDR to be grafted is added to primers for synthesizing a human antibody FR. Primers are prepared for each of the four FRs. It is generally considered that when grafting a mouse CDR to a human FR, selecting a human FR that has high identity to a mouse FR is advantageous for maintaining the CDR function. That is, it is generally preferable to use a human FR comprising an amino acid sequence which has high identity to the amino acid sequence of the FR adjacent to the mouse CDR to be grafted.
(28) The term bispecific means that the antibody is able to specifically bind to at least two distinct antigenic determinants, for example two binding sites each formed by a pair of an antibody heavy chain variable domain (VH) and an antibody light chain variable domain (VL) binding to different antigens or to different epitopes on the same antigen. Such a bispecific antibody is referred to as a 1+1 format. Other bispecific antibody formats are 2+1 formats (comprising two binding sites for a first antigen or epitope and one binding site for a second antigen or epitope) or 2+2 formats (comprising two binding sites for a first antigen or epitope and two binding sites for a second antigen or epitope). Typically, a bispecific antibody comprises two antigen binding sites, each of which is specific for a different antigenic determinant. The term valent as used within the current application denotes the presence of a specified number of binding domains in an antigen binding molecule. As such, the terms bivalent, tetravalent, and hexavalent denote the presence of two binding domains, four binding domains, and six binding domains, respectively, in an antigen binding molecule. The bispecific antibodies according to the disclosure are at least bivalent and may be trivalent or multivalent (e.g., tetravalent or hexavalent). In a particular aspect, the antibodies of the present disclosure have two or more binding sites and are bispecific. That is, the antibodies may be bispecific even in cases where there are more than two binding sites (i.e. that the antibody is trivalent or multivalent). The terms full length antibody, intact antibody, and whole antibody are used herein interchangeably to refer to an antibody having a structure substantially similar to a native antibody structure. Native antibodies refer to naturally occurring immunoglobulin molecules with varying structures. For example, native IgG-class antibodies are hetero-tetrameric glycoproteins of about 150,000 daltons, composed of two light chains and two heavy chains that are disulfide-bonded. From N- to C-terminus, each heavy chain has a variable region (VH), also called a variable heavy domain or a heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3), also called a heavy chain constant region. Similarly, from N- to C-terminus, each light chain has a variable region (VL), also called a variable light domain or a light chain variable domain, followed by a light chain constant domain (CL), also called a light chain constant region. The heavy chain of an antibody may be assigned to one of five types, called (IgA), (IgD), (IgE), (IgG), or (IgM), some of which may be further divided into subtypes, e.g., 1 (IgG1), 2 (IgG2), 3 (IgG3), 4 (IgG4), 1 (IgA1) and 2 (IgA2). The light chain of an antibody may be assigned to one of two types, called kappa () and lambda (), based on the amino acid sequence of its constant domain. An antibody fragment refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include but are not limited to Fv, Fab, Fab, Fab-SH, F(ab)2; diabodies, triabodies, tetrabodies, cross-Fab fragments; linear antibodies; single-chain antibody molecules (e.g., scFv); multispecific antibodies formed from antibody fragments and single domain antibodies. Single-domain antibodies are antibody fragments comprising all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In certain embodiments, a single-domain antibody is a human single-domain antibody. In addition, antibody fragments comprise single chain polypeptides having the characteristics of a VH domain, namely being able to assemble together with a VL domain, or of a VL domain, namely being able to assemble together with a VH domain to a functional antigen binding site and thereby providing the antigen binding property of full length antibodies. Antibody fragments can be made by various techniques, including but not limited to proteolytic digestion of an intact antibody as well as production by recombinant host cells (e.g., E. coli or phage), as described herein. Papain digestion of intact antibodies produces two identical antigen-binding fragments, called Fab fragments, each containing the heavy- and light-chain variable domains and also the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. As used herein, the term Fab fragment refers to an antibody fragment comprising a light chain fragment comprising a VL domain and a constant domain of a light chain (CL), and a VH domain and a first constant domain (CH1) of a heavy chain. Fab fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain including one or more cysteines from the antibody hinge region. Fab-SH are Fab fragments wherein the cysteine residue(s) of the constant domains bear a free thiol group. Pepsin treatment yields an F(ab)2 fragment that has two antigen-combining sites (two Fab fragments) and a part of the Fc region.
(29) A single-chain variable fragment (scFv) is a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of an antibody, connected with a short linker peptide of ten to about 25 amino acids. The linker is usually rich in glycine for flexibility, as well as serine or threonine for solubility, and can either connect the N-terminus of the VH with the C-terminus of the VL, or vice versa. This protein retains the specificity of the original antibody, despite removal of the constant regions and the introduction of the linker. In addition, antibody fragments comprising single chain polypeptides have the characteristics of a VH domain, namely being able to assemble together with a VL domain, or of a VL domain, namely being able to assemble together with a VH domain to a functional antigen binding site, thereby providing the antigen binding properties of full length antibodies.
(30) By specific binding it is meant that the binding is selective for the antigen and can be distinguished from unwanted or non-specific interactions with substrates other than the antigen. The ability of an antigen binding molecule to bind to a specific antigen can be measured either through an enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to one of skill in the ar, and traditional binding assays. In one embodiment of the present application, the extent of binding of an antigen binding molecule to an unrelated protein is less than about 10% of the binding of the antigen binding molecule to the antigen as measured, e.g., by SPR. In certain embodiments, a molecule that binds to the antigen has a dissociation constant (Kd) of <1 M, <100 nM, <10 nM, <1 nM, <0.1 nM, <0.01 nM, or <0.001 nM (e.g., 10.sup.7 M or less, e.g., from 10.sup.7 M to 10.sup.13 M, e.g., from 10.sup.9 M to 10.sup.13 M).
(31) Affinity or binding affinity refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, binding affinity refers to intrinsic binding affinity which reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (K.sub.d), which is the ratio of dissociation and association rate constants (k.sub.off and k.sub.on, respectively). Thus, equivalent affinities may comprise different rate constants, as long as the ratio of the rate constants remains the same. Affinity can be measured by common methods known in the art, including those described herein. A particular method for measuring affinity is Surface Plasmon Resonance (SPR). As used herein, the term high affinity of an antibody refers to an antibody having a K.sub.d of 10.sup.9 M or less and even more particularly 10.sup.19 M or less for a target antigen. The term low affinity of an antibody refers to an antibody having a K.sub.d of 10.sup.8 or higher.
(32) The term variable region or variable domain refers to the domain of an antibody heavy or light chain that is involved in binding the antigen binding molecule to antigen. The variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs). A single VH or VL domain may be sufficient to confer antigen-binding specificity.
(33) Hypervariable regions (HVRs) are also referred to as complementarity determining regions (CDRs), and these terms are used herein interchangeably in reference to portions of the variable region that form the antigen binding regions. This particular region has been described by Kabat et al., U.S. Dept. of Health and Human Services, where the definitions include overlapping or subsets of amino acid residues when compared against each other. Nevertheless, application of either definition to refer to a CDR of an antibody or variants thereof is intended to be within the scope of the term as defined and used herein. The appropriate amino acid residues which encompass the CDRs as defined by each of the above cited references are set forth below in Table A as a comparison. The exact residue numbers which encompass a particular CDR will vary depending on the sequence and size of the CDR. Those skilled in the art can routinely determine which residues comprise a particular CDR given the variable region amino acid sequence of the antibody.
(34) Kabat et al. also defined a numbering system for variable region sequences that is applicable to any antibody. One of ordinary skill in the art can unambiguously assign this system of Kabat numbering to any variable region sequence, without reliance on any experimental data beyond the sequence itself. As used herein, Kabat numbering refers to the numbering system set forth by Kabat et al., U.S. Dept. of Health and Human Services. Unless otherwise specified, references to the numbering of specific amino acid residue positions in an antibody variable region herein are made according to the Kabat numbering system. With the exception of CDR1 in VH, CDRs generally comprise the amino acid residues that form the hypervariable loops. CDRs also comprise specificity determining residues, or SDRs, which are residues that contact antigen. SDRs are contained within regions of the CDRs called abbreviated-CDRs, or a-CDRs. Exemplary a-CDRs (a-CDR-L1, a-CDR-L2, a-CDR-L3, a-CDR-H1, a-CDR-H2, and a-CDR-H3) occur at amino acid residues 31-34 of LI, 50-55 of L2, 89-96 of L3, 31-35B of HI, 50-58 of H2, and 95-102 of H3.) Unless otherwise indicated, HVR residues and other residues in the variable domain (e.g., FR residues) are numbered herein according to Kabat et al.
(35) By fused to or connected to is meant that the components (e.g., an antigen binding domain and a FC domain) are linked by peptide bonds, either directly or via one or more peptide linkers.
(36) The terms host cell, host cell line, and host cell culture are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include transformants and transformed cells, which include the primary transformed cell and progeny derived therefrom without regard to the number of passages.
(37) A therapeutically effective amount of an agent, e.g., a pharmaceutical composition, refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result. A therapeutically effective amount of an agent for example eliminates, decreases, delays, minimizes or prevents adverse effects of a disease.
(38) An individual or subject is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). Particularly, the individual or subject is a human. The term pharmaceutical composition refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered. A pharmaceutically acceptable excipient refers to an ingredient in a pharmaceutical composition, other than an active ingredient, which is nontoxic to a subject. A pharmaceutically acceptable excipient includes, but is not limited to, a buffer, a stabilizer, or a preservative.
(39) As used herein, treatment (and grammatical variations thereof such as treat or treating) refers to clinical intervention in an attempt to alter the natural course of the individual being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. In some embodiments, the molecules of the application are used to delay development of a disease or to slow the progression of a disease.
(40) The term cancer as used herein refers to proliferative diseases, such as lymphomas, lymphocytic leukemias, lung cancer, non-small cell lung (NSCL) cancer, bronchioloalviolar cell lung cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, gastric cancer, colon cancer, breast cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's Disease, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, prostate cancer, cancer of the bladder, cancer of the kidney or ureter, renal cell carcinoma, carcinoma of the renal pelvis, mesothelioma, hepatocellular cancer, biliary cancer, neoplasms of the central nervous system (CNS), spinal axis tumors, brain stem glioma, glioblastoma multiforme, astrocytomas, schwanomas, ependymonas, medulloblastomas, meningiomas, squamous cell carcinomas, pituitary adenoma and Ewings sarcoma, including refractory versions of any of the above cancers, or a combination of one or more of the above cancers. The cells used for measurement of cytotoxic activity may be the desired GPC3-expressing cells or a desired tissue containing these cells, for example, HepG2, PC-10 or NCI-H446 which are GPC3-expressing human cancer cell lines. GPC3 negative cells HPAC and LS-174T tumor cell lines are used herein as a control.
(41) The present application provides a novel GPC3 antibody or antigen binding fragment and related bispecific antibody or chimeric antigen receptor (CAR) with particularly advantageous properties such as high producibility, stability, binding affinity, biological activity, specific targeting of GPC3-positive cells, targeting efficiency, remaining tumor cell killing and reduced toxicity.
(42) The present application also relates to polynucleotides encoding the GPC3 antibody or antigen binding fragment, the bispecific antibody, or the chimeric antigen receptor (CAR) of the present application, and they may be inserted into discretionary expression vectors. Suitable hosts may be transformed with the expression vectors to produce cells that express the GPC3 antibody or antigen binding fragment, the bispecific antibody, or the chimeric antigen receptor (CAR) of the application. GPC3 antibody or antigen binding fragment, the bispecific antibody, or the chimeric antigen receptor (CAR) encoded by the polynucleotides can be obtained by culturing the cells that express the GPC3 antibody or antigen binding fragment, the bispecific antibody, or the chimeric antigen receptor (CAR). That is, the present application relates to vectors comprising a polynucleotide encoding a GPC3 antibody or antigen binding fragment, the bispecific antibody, or the chimeric antigen receptor (CAR) of the present application, cells carrying such a vector, and methods for producing GPC3 antibody or antigen binding fragment, the bispecific antibody, or the chimeric antigen receptor (CAR) of the application, which comprises culturing the cells and collecting GPC3 antibody or antigen binding fragment or the bispecific antibody from culture supernatants or collecting cells expressing the chimeric antigen receptor(CAR). These can be obtained by techniques similar to those for recombinant antibodies mentioned above.
Definitions
(43) The following is an explanation of the nomenclature of dual functional (bispecific) antibodies in the different formats generated as used herein: GPC3/CD3 bispecific antibody: (bispecific antibody against human Glypican 3 and CD3) GPC3/CD3-2 clone (bispecific antibody chimeric 2 against human Glypican 3 and CD3) GPC3/CD3-40 clone (bispecific antibody chimeric 40 against human Glypican 3 and CD3) GPC3/CD3-74 clone (bispecific antibody chimeric 74 against human Glypican 3 and CD3) GPC3/CD3-109 clone (bispecific antibody chimeric 109 against human Glypican 3 and CD3) GPC3/CD3-182 clone (bispecific antibody chimeric 182 against human Glypican 3 and CD3) GPC3 H1 (Humanized Heavy Chain candidate of rabbit Clone 40 Heavy chain against human Glypican 3) GPC3 H3 (Humanized Heavy Chain candidate of rabbit Clone 40 Heavy chain against human Glypican 3) GPC3 L1 (Humanized Light chain candidate of rabbit Clone 40 Light chain against human Glypican 3) GPC3 L3 (Humanized Light chain candidate of rabbit Clone 40 Light chain against human Glypican 3) GPC3 H1L1/CD3 (GPC3/CD3 humanized bispecific antibody of rabbit clone 40 against human Glypican 3 and CD3) GPC3 H1L1/CD3OPT1a3b (GPC3/CD3 bispecific antibody candidate of rabbit clone 40 against human Glypican 3 and CD3) GPC3 H1L1/CD3OPT1a3b2b1 (GPC3/CD3 bispecific antibody candidate of rabbit clone 40 against human Glypican 3 and CD3) GPC3 H1L3/CD3 (GPC3/CD3 bispecific antibody candidate of rabbit clone 40 against human Glypican 3 and CD3) GPC3 H1L3/CD3OPT1a3b (GPC3/CD3 bispecific antibody candidate of rabbit clone 40 against human Glypican 3 and CD3) GPC3 H1L3/CD3OPT1a3b2b1 (GPC3/CD3 bispecific antibody candidate of rabbit clone 40 against human Glypican 3 and CD3) GPC3 H3L3/CD3 (GPC3/CD3 bispecific antibody candidate of rabbit clone 40 against human Glypican 3 and CD3) GPC3 H3L3/CD3OPT1a3b (GPC3/CD3 bispecific antibody candidate of rabbit clone 40 against human Glypican 3 and CD3) GPC3 H3L3/CD3OPT1a3b2b1 (GPC3/CD3 bispecific antibody candidate of rabbit clone 40 against human Glypican 3 and CD3)
EXAMPLES
Example 1. Generation and Identification of Anti-Human GPC3 Monoclonal Antibodies
1. Generation of Anti-Human GPC3 Monoclonal Antibodies
(44) 1) Human GPC3 protein: A full length human GPC3 protein consists of 580 amino acids (GeneBank accession no. AFM30911.1), with two heparan sulfate (HS) side chains attached close to the C-terminal portion. Cleavage by furin between Arg358 and Cys359 of the full length human GPC3 protein by furin results in a 40-kDa N-terminal subunit and a 30-kDa C-terminal subunit (i.e. C-terminal fragment)linked by a disulfide bond. The C-terminal of the full length human GPC3 protein is close to a cell membrane (i.e. anchored via GPI to a cell membrane), and binding of an antibody to the membrane proximal region will help T cell engager to improve the antibody's killing activity.
(45) 2) Obtaining Anti-Human GPC3 Monoclonal Antibodies
(46) Three New England White rabbits were immunized with the full length human GPC3 protein, and antibodies against the membrane-anchored region (i.e. C-terminal of the human GPC3 protein) are screened with the C-terminal of the full length human GPC3 protein (e.g. the C-terminal fragment or C-terminal subunit).
(47) ELISA assay was performed to assess the immune response of the 3 rabbits against the full length human GPC3 protein as well as the C-terminal of full length human GPC3 protein. Two immunized rabbits that have the optimal titer against the full length human GPC3 protein as well as the C-terminal of GPC3 protein were chosen for the development of anti-human GPC3 monoclonal antibodies.
(48) PBMCs were isolated from the two immunized rabbits that have the optimal titer against GPC3, and antigen-specific B cells were enriched via density gradient centrifugation using Ficoll-Pague, then the antigen-specific B cells were differentiated to plasma cells for a 5 to 7-day culture. ELISA assay was performed to identify clones that were selectively positive to C-terminal of full length human GPC3 protein. A total of 117 clones were recovered from recombinant expression. Among the 117 clones, the top 15 clones with strongest binding affinity to human GPC3 proteins and cynomolgus monkey GPC3 protein were selected for further testing. The VH and VL sequences of the above said top 15 clones are shown in Table 1.
(49) TABLE-US-00001 TABLE1 TheVHorVLsequenceofthetop15clones Clone Vdomain Sequence 17 HeavyChain QCQSVEESGGRLVTPGTPLTLTCTVSGFSLSTYDMSWVRQAPGKGLEYIGWIN SGGSPYYARWAKGRFTISKTSSTTVDLKMTSPTTEDTATYFCARHRSGYYGDI WGPGTLVTVSL(SEQIDNO.37) 17 LightChain DPVLTQTPSSVSAAVGDTVSINCQSSQNVYKNRLAWYQQKPGQPPKLLIYGA STLASGVPSRFKGSGSGTQFTLTISDLECDDAATYYCAGGYSTIVDNTFGGGT EVVVK(SEQIDNO.38) 23 HeavyChain QCQSVEESGGRLVTPGTALTLTCTVSGFSLSTYWMTWVRQAPGKGLEWIGIIN PGGSAYYASWAKGRFTISKTSSATVDLKMTSLTAADTATYFCAGGGGMDPW GPGTLVTVSS(SEQIDNO.39) 23 LightChain DPVLTQTPSSVSAAVGGTVSISCQSSQSIIKNYLSWFQHKPGQPPKRLIYRASTL PSGVPSRFEGSGSGTEFTLTISDLECDDAATYYCAASYSDNIYVFGGGTEVVV K(SEQIDNO.40) 41 HeavyChain HCQSVEESGGRLVTPGTALTLTCTVSGFSLSTYWMTWVRQAPGKGLEWIGIIN PGGSAYYASWAKGRFTISKASSATVDLKMTSLTAADTATYFCAGGGGMDPW GPGTLVTVSS(SEQIDNO.41) 41 LightChain DPVLTQTPSSVSAAVGGTVSISCQSSQSIIKNYLSWFQHKPGQPPKRLIYRASTL PSGVPSRFEGSGSGTEFTLTINDLECDDAATYYCAASYSDNIYVFGGGTEVVV K(SEQIDNO.42) 8 HeavyChain QCQSVEESGGRLVTPGTALTLTCTVSGFSLSTYWMTWVRQAPGKGLEWIGIIN PGGSAYYASWAKGRFTISKTSSATVDLKMTSLTAADTATYFCAGGGGMDPW GPGTLVTVSS(SEQIDNO.43) 8 LightChain DPVLTQTPSSVSAAVGGTVSISCQSSQSIIKNYLSWFQHKPGQPPKRLIYRASTL PSGVPSRFEGSGSGTEFTLTISDLECDAATYYCAASYSDNIYVFGGGTEVVV K(SEQIDNO.44) 12 HeavyChain QCQSVEESGGRLVTPGTPLTLTCTVSGFSLSTYWMTWVRQAPGKGLEWIGIIN PSGSAFYASWAKGRFTISKTSSATVDLKMTSLTAADTATYFCAGGGGMDPW GPGTLVTVSS(SEQIDNO.45) 12 LightChain DPVLTQTPSSVSAAVGGTVSISCQSSQSIVKNYLSWFQQKPGQPPKRLIYKAST LPSGVPSRFKGSGSGTEFTLTISDLECDDAATYYCAASYSDNIYVFGGGTEVV VK(SEQIDNO.46) 22 HeavyChain QCQSVEESGGRLVTPGTPLTLTCTVSGFSLSTYWMTWVRQAPGKGLEWIGIIN PTGRAYYASWAKGRFTISKTSSATVDLKMTSLTAADTATYFCAGGGGMDPW GPGTLVTVSS(SEQIDNO.47) 22 LightChain DPVLTQTPSSVSAAVGGTVTINCQSSQSVYSNYLSWFQKKPGQPPKRLIYKAS TLVSGVPSRFVGSGSGTEFTLTISDLECDDAATYYCAASYSGNIYVFGGGTEV VVK(SEQIDNO.48) 40 HeavyChain QCQSVEESGGRLVTPGTPLTLTCTVSGFSLSTYWMTWVRQAPGKGLEWIGIIS PAGSAYYASWAKGRFTISKTSSATVDLKMTSLTTADTATYFCAGGGGMDPW GPGTLVTVSS(SEQIDNO.16) 40 LightChain DPVLTQTPSSVSAAVGGTVSISCQSSQSIVKNYLSWFQQKPGQPPKRLIYKAST LPSGVPSRFKGSGSGTEFTLTISDLECDDAATYYCAASYSDNIYVFGGGTEVV VK(SEQIDNO.17) 49 HeavyChain QCQSVEESGGRLVTPGTPLTLTCTVSGFSLSTYWMTWVRQAPGKGLEWIGIIN PSGSAYYASWAKGRFTISKTSSATVDLKMTSLTAADTATYFCAGGGGMDPW GPGTLVTVSS(SEQIDNO.49) 49 LightChain DPVLTQTPSSVSAAVGGTVSISCQSSQSIVKNYLSWFQQKPGQPPKRLIYKAST LPSGVPSRFKGSGSGTEFTLTISDLECDDAATYYCAASYSDNIYVFGGGTEVV VK(SEQIDNO.50) 74 HeavyChain QCQSVEESGGRLVTPGTPLTLTYTVSGFSLNNYDMSWVRQAPGKGLQYIGWI NSGGTAYYASWAKGRFTISKTSSTTVDLKMTSPTTEDTATYFCARHRYGYYG DIWGPGTLVTVSL(SEQIDNO.51) 74 LightChain DPVLTQTPSSVSAAVGGTVTINCQSSQNVYNNNRLAWYQQKLGQPPKLLIYF ASKLASGVPSRFSGSGSGTQFTLTISGVQCDDAATYYCAGGYNTIVDNGFGGG TEVVVK(SEQIDNO.52) 109 HeavyChain QCQSVEESGGRLVTPGTPLTLTYTVSGFSLSSYDMSWVRQAPGKGLQYIGWM NTNGSAYYATWAKGRFTISKTSSTTVDLKMTSPTTEDTATYFCARHRSGYY (SEQIDNO.53) 109 LightChain DPVLTQTPSSVSAAVGGTVTINCQSSQNVLNQNRLAWYQQKPGQPPKLLIYW ASKLASGVPSRFKGSGSGTQFTLTISGVQCDDAATYYCAGGYSSIVYNAFGGG TEVVVK(SEQIDNO.54) 121 HeavyChain QCQSVEESGGRLVTPGTPLTLTCTVSGFSLSTYWMTWVRQAPGKGLEWIGIIN PAGSAYYASWAKGRFTISKTSSATVDLKMTSLTAVDTATYFCAGGGGMDPW GPGTLVTVSS(SEQIDNO.55) 121 LightChain DPVLTQTPSSVSAAVGGTVTISCQSSQNIIKDYLSWFQQKPGQPPKRLIYKTST LPSGVPSRFKGSGSGTEFTLTISDLECDDAATYYCAASYSGNIYVFGGGTEVV VK(SEQIDNO.56) 122 HeavyChain QCQSVEESGGRLVTPGTPLTLTYTVSGFSLSNYDMSWVRQAPGKGLEYIGWI NTGGSVYYASWAKGRFTISKTSSTTVDLKLTSPTTEDTATYFCARHRSGYFGD IWGPGTLVTVSL(SEQIDNO.57) 122 LightChain DPVLTQTPSSVSAAVGGTVTINCQSSQNVYNNNRLAWYQQKPGQPPKLLIYF ASKLASGVPSRFKGNGSGTQFTLTISGVQCDDAATYYCAGGYNSIVDNGFGG GTEVVVE(SEQIDNO.58) 182 HeavyChain QCQSVEESGGRLVTPGTPLTLTYTVSGFSLSNYDMSWVRQAPGKGLEYIGWI NTGGSVYYASWAKGRFTISKTSSTTVDLKLTSPTTEDTATYFCARHRSGYFGD IWGPGTLVTVSL(SEQIDNO.59) 182 LightChain DPVLTQTPSSVSAAVGGTVTINCQSSQNVYNNNRLAWYQQKPGQPPKLLIYF ASKLASGVPSRFKGNGSGTQFTLTISGVQCDDAATYYCAGGYNSIVDNGFGA GTEVVVK(SEQIDNO.60) 2 HeavyChain QCQSVEESGGRLVTPGTPLTLTCTVSGFSLSTYNMGWIRQAPGEGLEYIGTISS STGNTYYATWAKGRFTISKTSSTTVDLKITNLTTEDTATYFCVRVNNMGDIW GPGTLVTVSL(SEQIDNO.27) 2 LightChain AIVMTQTPSSKSVAVGDTVTINCQASESVYKDNRLAWFQQKPGQAPKLLIYL ASTLASGVPSRFKGSGSGTEFTLTISDVVCDDAATYYCGGYKDSLFDGFPFGG GTEVVVK(SEQIDNO.28) 5 HeavyChain QCQSVEESGGRLVTPGTPLTLTCTVSGFSLSTYNMGWVRQAPGEGLEYIGTIS SSTGNTYYATWAKGRFTISKTSSTTVDLKITNLTTEDTATYFCVRVNNMG DIWGPGTLVTVSL(SEQIDNO.61) 5 LightChain AIVMTQTPSSKSVAVGDTVTINCQASESVYKDNRLAWFQQKPGQPPKLLIYLA STLASGVPSRFKGSGSGTEFTLTISDVVCDDAATYYCGGYKDSLFDGFPFGGG TEVVVK(SEQIDNO.62)
2. Identification of Anti-Human GPC3 IgG1 Antibodies
(50) The Rabbit CL, CH1, CH2 and CH3 sequences were replaced with human CL, CH1, CH2 and CH3 sequences in the rabbit anti-human GPC3 clones to obtain anti-human GPC3 IgG1 antibodies. Sequences of CL, CH1, CH2, CH3 and Hinge regions for anti-human GPC3 IgG1 antibodies are shown in table 2.
(51) TABLE-US-00002 TABLE2 SequencesofCL,CH1,CH2,CH3andHingeregionsforanti- humanGPC3IgG1antibodies CLofhuman RTVAAPSVFIFPPSDEQLKSGTASVVCLLN NFYPREAKVQWKVDNALQSGNSQESVTE QDSKDSTYSLSSTLTLSKADYEKHKVYAC EVTHQGLSSPVTKSFNRGECTS(SEQIDNO. 63) CH1ofhuman ASTKGPSVFPLAPSSKSTSGGTAALGCLVK DYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQTYICNVNHKPS NTKVDKKVEPKSC(SEQIDNO.64) Hinge EPKSCDKTHTCPPCP(SEQIDNO.65) CH2ofhumanIgG1 APEAAGGPSVFLFPPKPKDTLMISRTPEVT heavychain(wildtype) CVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNG KEYKCKVSNKALPAPIEKTISKAK(SEQID NO.15) CH3ofhumanIgG1 GQPREPQVYTLPPSREEMTKNQVSLTCLV heavychain KGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK(SEQIDNO. 66)
(52) 1) Antigen binding affinity of the top 15 clones (i.e. 15 anti-human GPC3 IgG1 antibodies) was assessed by Biacore assays (data shown in Table 3).
(53) In general, full length human GPC3 His was immobilized on CMS sensor chip using amine coupling method followed by anti-human GPC3 IgG1 antibodies on CMS chip in HBS-EP buffer at a concentration ranging from 1 g/mL to 0.125 g/mL. Binding affinity was determined by SPR using a Biacore T200 instrument.
(54) TABLE-US-00003 TABLE 3 Antigen binding affinity of the top 15 clones was assessed by Biacore assays Ranking Ka Kd KD (Excel) Antibodies (a/s) (1/s) (nM) RuMax 1 74 1.0 10.sup.7 3.4 10.sup.4 0.02 24.32 2 2 8.0 10.sup.5 3.9 10.sup.4 0.00004 2.0 3 5 4 182 1.0 10.sup.7 1.9 10.sup.3 0.02 24.32 5 109 2.2 10.sup.6 9.3 10.sup.4 0.4 11.62 6 122 9.1 10.sup.6 1.8 10.sup.3 0.2 17.32 7 17 1.7 10.sup.7 3.7 10.sup.3 0.2 10.74 8 8 3.5 10.sup.6 4.2 10.sup.4 0.1 3.7 9 40 2.7 10.sup.6 1.8 10.sup.3 0.6 2.4 10 23 11 41 2.4 10.sup.6 3.2 10.sup.3 1.3 1.49 12 12 2.5 10.sup.6 1.6 10.sup.3 0.6 1.4 13 22 3.1 10.sup.6 5.2 10.sup.4 1.6 2.9 14 49 1.7 10.sup.6 1.7 10.sup.3 0.9 1.7 15 121 2.9 10.sup.6 1.5 10.sup.3 0.5 2.0
(55) 2) HepG2 cell surface binding affinity of the top 15 clones (i.e. 15 anti-human GPC3 IgG1 antibodies) was assessed by flow cytometry (data shown in Table 4).
(56) A HepG2 cell line expressing full length human GPC3 was used for flow cytometry analysis. In general, after dissociating cells and washing in PBS, 110.sup.5 target cells were seeded in a 96 well plate. The anti-GPC3 IgG1 antibodies prepared in a final concentration of 25 g/mL were incubated with cells for 1 hour at 4 C. After washing with FACS wash buffer, plates were incubated with PE conjugated goat anti-Human IgG, Fc Fragment Specific antibody (1:200 diluted in FACS wash buffer) for 20 minutes at 4 C. Mean fluorescence intensity (MFI) was measured using NovoCyte 2060 and results were analyzed by GraphPad software.
(57) TABLE-US-00004 TABLE 4 Assessment of HepG2 cell surface binding affinity of the top 15 clones by flow cytometry HepG2 Cell binding EC50 Ranking Antibodies (Fold ratio) (nM) 1 74 142.62 0.0042 2 2 693.58 0.0076 3 5 4 182 5 109 89.1 0.0045 6 122 128.99 0.0053 7 17 39.85 0.043 8 8 79.5 0.012 9 40 103.94 0.013 11 41 116.12 0.014 12 12 124.88 0.014 13 22 120.58 0.013 14 49 114.66 0.012 15 121 74.57 0.0061
(58) 3) Confirmation of binding specificity to GPC3 proteins (i.e. C-terminal fragment) of anti-GPC3 antibody by ELISA.
(59) The N-terminal of the GPC3 protein can be shed and is present in circulation. Therefore, the antibody of interest needs to target the C-terminal of the GPC3 protein and avoid binding to the soluble N-terminal fragment. The GPC3 binding epitopes of the top 15 clones (i.e. 15 anti-human GPC3 IgG1 antibodies) were determined by their competitive binding to benchmark antibody GC33 (which is known to bind to the C-terminal of the GPC3 protein, see US20180244805A1 for reference), the results are shown in Table 5, Table 5 shows all 15 Clones are specificity binding to the C-terminal fragment of the full length human GPC3 protein, and binding epitopes of the clones are non-overlapped, overlapped and partially overlapped with that of GC33.
(60) TABLE-US-00005 TABLE 5 The GPC3 binding epitopes of the top clones. The clones that binds to (A) Non-overlapped (C) Partially overlapped epitopes with GC33 (B) Same epitopes as GC33 epitopes with GC33 Indications Positive binding to GPC3 Negative binding to GPC3 antigen on GC33-coated antigen on GC33-coated Inconsistent binding to ELISA and individual Ab- ELISA and individual Ab- GPC3 in all repeating coated ELISA coated ELISA binning experiments Clone 17 41 22 ID 109 8 40 122 12 49 121 74 182 2 5
(61) Based on the above results from affinity ranking, cell surface binding, binding specificity, binding epitope and sequence analysis, the top 5 candidates (Clone 2, Clone 40, Clone 74, Clone 109, and Clone 182) were selected for the development of GPC3/CD3 bispecific antibody.
Example 2. Generation of Rabbit-Human Chimeric GPC3/CD3 Bispecific Antibody
(62) The symmetric molecule structure of bispecific antibody was chosen for the format of GPC3/CD3 bispecific antibody development. A schematic representation of GPC3/CD3 bispecific antibody structure of one or more embodiments of the present application is shown in
(63) Further, to avoid Ig Fc mediated-ADCC/CDC function, LALA mutation (L234A, L235A) was incorporated in the human IgG1 Fc region to eliminate effector functions in the present application. The position of the mutations in IgG1 Fc region of the present application are in Eu Numbering.
(64) The VH and VL sequences of the 5 Chimeric bispecific antibodies selected for testing are shown in Table 6. Sequences of CL, CH1, CH2, CH3 and Hinge regions for the 5 Chimeric bispecific antibodies selected are shown in Table 7.
(65) TABLE-US-00006 TABLE6 VHandVLsequencesofthe5Chimericbispecificantibodiesselected(Underlined SequencesrepresentCDRs,theanalysissystemisKabatsystem) anti-GPC3 anti-CD3(wild-type) BsAb VH VL VH VL GPC3/CD3-2 QCQSVEESGGRLVTPGT AIVMTQTPSSKSVAVGDTV EVQLVESGGG QTVVTQEPSL PLTLTCTVSGFSLSTYN TINCQASESVYKDNRLAW LVQPGGSLKL TVSPGGTVTL MGWIRQAPGEGLEYIGT FQQKPGQAPKLLIYLASTL SCAASGFTFN TCGSSTGAVT ISSSTGNTYYATWAKGR ASGVPSRFKGSGSGTEFTL KYAMNWVR SGYYPNWVQ FTISKTSSTTVDLKITNL TISDVVCDDAATYYCGGY QAPGKGLEW QKPGQAPRGL TTEDTATYFCVRVNNM KDSLFDGFPFGGGTEVVV VARIRSKYNN IGGTKFLAPG GDIWGPGTLVTVSL K(SEQIDNO.28) YATYYADSV TPARFSGSLL (SEQIDNO.27) CDRs: KDRFTISRDD GGKAALTLSG CDRs: QASESVYKDNRLA(SEQID SKNTAYLQM VQPEDEAEYY STYNMG(SEQIDNO. NO.24) NNLKTEDTA CALWYSNRW 21) LASTLAS(SEQIDNO.25) VYYCVRHGN VFGGGTKLTV TISSSTGNTYYATWAKG GGYKDSLFDGFP(SEQID FGNSYISYWA L (SEQIDNO.22) NO.26) YWGQGTLVT (SEQIDNO. VNNMGDI(SEQIDNO. VSS 11) 23) (SEQIDNO. 10) GPC3/CD3- QCQSVEESGGRLVTPGT DPVLTQTPSSVSAAVGGTV 40 PLTLTCTVSGFSLSTYW SISCQSSQSIVKNYLSWFQ MTWVRQAPGKGLEWIG QKPGQPPKRLIYKASTLPS IISPAGSAYYASWAKGR GVPSRFKGSGSGTEFTLTIS FTISKTSSATVDLKMTS DLECDDAATYYCAASYSD LTTADTATYFCAGGGG NIYVFGGGTEVVVK(SEQ MDPWGPGTLVTVSS IDNO.17) (SEQIDNO.16) CDRs: CDRs: QSSQSIVKNYLS(SEQID GFSLSTYWMT(SEQID NO.4) NO.1) KASTLPS(SEQIDNO.5) IISPAGSAYYASWAKG AASYSDNIYV(SEQID (SEQIDNO.2) NO.6) AGGGGMDP(SEQID NO.3) GPC3/CD3- QCQSVEESGGRLVTPGT DPVLTQTPSSVSAAVGGTV 74 PLTLTYTVSGFSLNNYD TINCQSSQNVYNNNRLAW MSWVRQAPGKGLQYIG YQQKLGQPPKLLIYFASKL WINSGGTAYYASWAKG ASGVPSRFSGSGSGTQFTL RFTISKTSSTTVDLKMTS TISGVQCDDAATYYCAGG PTTEDTATYFCARHRYG YNTIVDNGFGGGTEVVVK YYGDIWGPGTLVTVSL (SEQIDNO.68) (SEQIDNO.67) GPC3/CD3- QCQSVEESGGRLVTPGT DPVLTQTPSSVSAAVGGTV 109 PLTLTYTVSGFSLSSYD TINCQSSQNVLNQNRLAW MSWVRQAPGKGLQYIG YQQKPGQPPKLLIYWASK WMNTNGSAYYATWAK LASGVPSRFKGSGSGTQFT GRFTISKTSSTTVDLKM LTISGVQCDDAATYYCAG TSPTTEDTATYFCARHR GYSSIVYNAFGGGTEVVV SGYY(SEQIDNO.69) K(SEQIDNO.70) GPC3/CD3- QCQSVEESGGRLVTPGT DPVLTQTPSSVSAAVGGTV 182 PLTLTYTVSGFSLSNYD TINCQSSQNVYNNNRLAW MSWVRQAPGKGLEYIG YQQKPGQPPKLLIYFASKL WINTGGSVYYASWAKG ASGVPSRFKGNGSGTQFTL RFTISKTSSTTVDLKLTS TISGVQCDDAATYYCAGG PTTEDTATYFCARHRSG YNSIVDNGFGAGTEVVVK YFGDIWGPGTLVTVSL (SEQIDNO.72) (SEQIDNO.71) Underline shows the CDRs
(66) TABLE-US-00007 TABLE7 SequencesofCL,CH1,CH2,CH3andHingeregionsforthe5 Chimericbispecificantibodiesselected CLofhuman RTVAAPSVFIFPPSDEQLKSGTASVVCLLN NFYPREAKVQWKVDNALQSGNSQESVTE QDSKDSTYSLSSTLTLSKADYEKHKVYAC EVTHQGLSSPVTKSFNRGECTS(SEQIDNO. 63) CH1ofhuman ASTKGPSVFPLAPSSKSTSGGTAALGCLVK DYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQTYICNVNHKPS NTKVDKKVEPKSC(SEQIDNO.64) Hinge EPKSCDKTHTCPPCP(SEQIDNO.65) CH2ofhumanIgG1 APELLGGPSVFLFPPKPKDTLMISRTPEVTC heavychain(withLALA VVVDVSHEDPEVKFNWYVDGVEVHNAKT mutation) KPREEQYNSTYRVVSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTISKAK(SEQID NO.13) CH3ofhumanIgG1 GQPREPQVYTLPPSREEMTKNQVSLTCLV heavychain KGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK(SEQIDNO. 66)
Example 3. Chimeric GPC3/CD3 Bispecific Antibody Mediates T Cell Killing of GPC3-Expressing Target Cells In Vitro
(67) To test whether GPC3/CD3 bispecific antibodies render T cells cytotoxic toward GPC3-positive targets, we performed standard cytotoxicity assays. PBMCs were pre-activated by culturing with anti-CD3 and anti-CD28 antibodies (stem cell technologies) in the presence of IL2 (20 ng/mL) for 6 days at E:T ratio of 10:1. The cytotoxicity (i.e. lysis activity) mediated by different GPC3/CD3 bispecific antibodies was assessed by Luciferase from live HepG2 Luc cells after 16 hour-incubation in a serial dilution of testing GPC3/CD3 bispecific antibodies. GPC3/CD3 bispecific antibodies (Clones GPC3/CD3-2 and GPC3/CD3-40) enhanced T-cell redirected cytotoxicity of GPC3-expressing HepG2 tumor cells in a dose-dependent manner. However, lower lysis to GPC3-expressing HepG2 tumor cells was observed in the treatment of GPC3/CD3-74, GPC3/CD3-109, and GPC3/CD3-182 (
Example 4. Chimeric GPC3/CD3 Bispecific Antibodies Induce Cytokines Release by PBMCs
(68) Cytokines secreted by activated T cells can profoundly affect immune responses in vitro and in vivo. Several cytokines limit tumor cell growth by direct anti-proliferative or pro-apoptotic activity, or indirectly by stimulating the cytotoxic activity of immune cells against tumor cells. IFN- and IL-2 play a very important role at the interface of innate and adaptive immune systems. INF- signaling increases the presence of antigenic peptides to T lymphocytes and increase antigen specific CD8+ T cell activation and T cell mediated tumor killing. To clearly determine the functional effects of GPC3/CD3 bispecific antibody on effector cytokines production in vitro, pre-activated T cells were cocultured with GPC3-expressing target cells in the presence of GPC3/CD3 bispecific antibodies at serial dilution for 16 hours. The supernatant was collected for cytokines assay. Similarly, IFN-, IL-2, and TNF- secretion markedly increased in GPC3/CD3-2 clone and GPC3/CD3-40 clone treated PBMCs (
Example 5. Triggering TCR by Chimeric GPC3/CD3 Bispecific Antibody Induces GPC3 Dependent-NFAT Activity in Jurkat Cells In Vitro
(69) The binding of antigens or anti-CD3 antibodies to T cell receptor initiates TCR/CD3-mediated signaling, which leads to activation of the NFAT pathway. Activating NFAT signaling drives T cell proliferation, cytokine production, and activation cell surface marker expression. To assess whether crosslinking of TCR by GPC3/CD3 bispecific antibodies can induce activation signaling on T cells, we used a classical TCR-NFAT-luciferase gene report system to perform NFAT activity assay. Three settings were designed to assess T cell immune response mediated by GPC3/CD3 antibodies (bispecific antibody with wild-type anti-CD3 sequences in table 6): a) adding GPC3/CD3 bispecific antibodies into coculture of Jurkat cells and GPC3-transfected HEK293 T cells; b) adding GPC3/CD3 bispecific antibodies into coculture of Jurkat cells and GPC3 negative SK-Hep-1 cells; and 3) adding isotype control/CD3 bispecific antibody (bispecific antibody with wild-type anti-CD3 sequences in table 6) as negative control into coculture of Jurkat cells and GPC3-transfected HEK293 T cells. As expected, a strong upregulation of the NFAT reporter gene activity was only observed in treatment of Jurkat cells with GPC3/CD3 bispecific antibody clone 2 and clone 40 in coculture with GPC3 transfected HEK293 T cells, however NFAT activity levels were low with GPC3/CD3 bispecific antibody clones 74, 109, and 182 (
(70) TABLE-US-00008 TABLE8 VHandVLsequencesofIC(isotypecontrol) antibody VH EVQLVQSGAEVKKSGESLKISCKGSGYSFTSY WIGWVRQMPGKGLEWMGIFYPGDSSTRYSPS FQGQVTISADKSVNTAYLQWSSLKASDTAMY YCARRRNWGNAFDIWGQGTMVTVSS(SEQID NO.73) VL EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYL AWYQQKPGQAPRLLIYGASSRATGIPDRFSGS GSGTDFTLTISRLEPEDFAVYYCQQYGSSTWTF GQGTKVEIK(SEQIDNO.74)
Example 6. Humanization of Lead Anti-GPC3 candidates, and Characterization of Top Humanized Anti-GPC3 Candidates and Humanized GPC3/CD3 Bispecific Antibody
(71) Humanization of two lead anti-GPC3 candidates (clone #40 and clone #2), selected based on better cytotoxicity and better cytokines readout, were performed. Sequences of rabbit anti-GPC3 antibodies were analyzed for homologous germ-line variable regions of database. Further antibodies were also optimized for optimal thermal stability and developability assessment. The humanized versions of the heavy and the light chain were transiently expressed in combinations to identify the antibody variants with best retention of antigen binding in vitro, thermostability and specific binding.
(72) Since chimeric antibodies could potentially elicit immunogenic responses in human patients, the lead chimeric anti-GPC3 clone 40 must be humanized via grafting the non-human complementarity-determining regions (CDRs) onto a human germline framework. As a result, three humanized light chains L1, L2, L3, and three humanized heavy chains H1, H2, H3, were generated through a grafting process. Two humanized light chains L1, L3, and two humanized heavy chains H1, H3, were chosen. The variable region sequences of the humanized anti-GPC3 clone 40 are shown in Table 9. Sequences of CL, CH1, CH2, CH3 and Hinge regions for the humanized anti-GPC3 clone 40 are shown in Table 7.
(73) TABLE-US-00009 TABLE9 Variableregionsequencesofthehumanizedanti-GPC3clone40(Underlined SequencesrepresentCDRs,theanalysissystemisKabatsystem) H1 QCQSVEESGGGLVQPGGSLRLSCAVSGFSLSTYWMTWVRQAPGKGLEWIGIISPAGSAY (VH1) YASWAKGRFTISRDNSATVYLQMNSLRAEDTAVYFCAGGGGMDPWGQGTLVTVSS (SEQIDNO.7) H2 QCQSVEESGGGLVQPGGSLRLSCAVSGFSLSTYWMTWVRQAPGKGLEWIGIISPAGSAY (VH2) YASWAKGRFTISKTNSATVYLQMNSLRAEDTATYFCAGGGGMDPWGQGTLVTVSS (SEQIDNO.18) H3 QCQSVEESGGGLVQPGGSLRLSCTVSGFSLSTYWMTWVRQAPGKGLEWIGIISPAGSAY (VH3) YASWAKGRFTISKTNSATVYLQMNSLRAEDTATYFCAGGGGMDPWGQGTLVTVSS (SEQIDNO.19) L1 DIVLTQSPSAMSASVGDRVTITCQSSQSIVKNYLSWFQQKPGKPPKRLIYKASTLPSGVPS (VL1) RFSGSGSGTEFTLTISSLQPEDFATYYCAASYSDNIYVFGGGTKVEIK(SEQIDNO.8) L2 DPVLTQSPSAMSASVGDRVTITCQSSQSIVKNYLSWFQQKPGKPPKRLIYKASTLPSGVP (VL2) SRFSGSGSGTEFTLTISSLQPEDFATYYCAASYSDNIYVFGGGTKVEIK(SEQIDNO.20) L3 DPVLTQSPSAMSASVGDRVTITCQSSQSIVKNYLSWFQQKPGQPPKRLIYKASTLPSGVP (VL3) SRFSGSGSGTEFTLTISSLQPEDFATYYCAASYSDNIYVFGGGTKVEIK(SEQIDNO.9) Underline shows the CDRs
(74) We further assessed whether the humanized versions of anti-GPC3 clone 40 were able to exhibit lysis activity as potent as seen previously in a chimeric form of anti-GPC3 clone 40 and so we compared in vitro cytotoxicity of humanized anti-GPC3 clone H1L1, H1L3 and H3L3 pairs in CD3 bispecific format (i.e. humanized GPC3/CD3 bispecific antibody with wild-type anti-CD3 sequences in table 6). The humanized H1L1/CD3, H1L3/CD3, and H3L3/CD3 bispecific antibodies were tested for redirecting T cell-mediated cytotoxicity against GPC3 positive and GPC3 negative tumor cell lines. HepG2 tumor cells were cocultured with stimulated human PBMCs at an effector/target ratio of 10:1 in a serial dilution of GPC3/CD3 BsAbs for 16 hours. The bispecific antibodies were found to induce cytolysis of GPC3 expressing HepG2 target cells in a dose dependent manner (
Example 7. Characterization of Humanized Anti-GPC3/CD3 Bispecific Antibodies
(75) To characterize humanized GPC3 antibody, humanized Heavy chains and Light chains were combined with various CD3 binders to generate humanized GPC3/CD3 bispecific antibodies. Important objectives of humanization are also the retention of high antigen binding affinity as well as preservation of preferred biophysical properties of the parental chimeric antibodies. The variable region sequences of various CD3 binders used herein are shown in Table 10.
(76) TABLE-US-00010 TABLE10 ThevariableregionsequencesofvariousCD3binders VH VL CD3 EVQLVESGGGLVQPGGSLKLSCAA QTVVTQEPSLTVSPGGTVTLTCG (wild-type) SGFTFNKYAMNWVRQAPGKGLE SSTGAVTSGYYPNWVQQKPGQA WVARIRSKYNNYATYYADSVKDR PRGLIGGTKFLAPGTPARFSGSLL FTISRDDSKNTAYLQMNNLKTEDT GGKAALTLSGVQPEDEAEYYCA AVYYCVRHGNFGNSYISYWAYWG LWYSNRWVFGGGTKLTVL QGTLVTVSS(SEQIDNO.10) (SEQIDNO.11) CD3OPT1a3b EVQLVESGGGLVKPGGSLKLSCAA QTVVTQEPSLTVSPGGTVTLTCG (mutant-type) SGFTFSTYAMNWVRQAPGKGLEW SSTGAVTSGYYPNWVQQKPGQA VARIRSKYNNYATYYADSVKDRFT PRGLIGGTKFLAPGTPARFSGSLL ISRDDSKNTAYLQMNNLRTEDTAV GGKAALTLSGVQPEDEAEYYCA YYCVRHGNWGNSYISYWAYWGQ LWYSNRWVFGGGTKLTVL GTTVTVSS(SEQIDNO.12) (SEQIDNO.11) CD3OPT1a3b2b1 EVQLVESGGGLVKPGGSLKLSCAA QTVVTQEPSLTVSPGGTVTLTCG (mutant-type) SGFTFSTYAMNWVRQAPGKGLEW SSTGAVTSGYYPNWVQQKPGQA VARIRSKYNNYATTYADSVKDRFTI PRGLIGGTKFLAPGTPARFSGSLL SRDDSKNTAYLQMNNLRTEDTAV GGKAALTLSGVQPEDEAEYYCA YYCVRHGNWGNSYISYWAYWGQ LWYSNRWVFGGGTKLTVL GTTVTVSS(SEQIDNO.14) (SEQIDNO.11)
(77) Based on our hypothesis that reducing affinity for either antigen in a bispecific antibody format may reduce cytokine release without necessarily reducing cytotoxic activity (i.e. lysis activity), various sets of anti-GPC3 and anti-CD3 arms having different affinities against each antigen were generated and produced in a bispecific antibody format. The resultant GPC3 bispecific antibodies were designated as H1L1/CD3, H1L1/CD3OPT1a3b, H1L1/CD3OPT1a3b2b1, H1L3/CD3, H1L3/CD3OPT1a3b, and H1L3/CD3OPT1a3b2b1. Each of the two light chains of anti-GPC3 (e.g., H1L1 or H1L3) is fused to a single chain variable fragment (scFv) of anti-CD3 (e.g., CD3, CD3OPT1a3b, or CD3OPT1a3b2b1) via a peptide linker GGGGSGGGGSGGGGS, to create two light chain fusion polypeptides.
(78) The binding affinities of humanized GPC3/CD3 bispecific antibodies to human GPC3 protein were measured by Biacore and cell binding assays. As shown in
(79) As T-cell bispecific antibodies may induce cytokine release syndrome and result in severe infusion-related reaction, the major challenge for T-cell bispecific antibody development is how to substantially reduce cytokine release but retain a significant cytotoxic activity.
(80) The resultant humanized GPC3/CD3 bispecific antibodies H1L1/CD3, H1L1/CD3OPT1a3b, H1L1/CD3OPT1a3b2b1, H1L3/CD3, H1L3/CD3OPT1a3b, and H1L3/CD3OPT1a3b2b1, in which OPT CD3 binders were fully characterized by cytotoxicity and cytokine release assays in the generation of our other (non-GPC3) bispecific antibody programs (see U.S. Ser. No. 62/974,744 or PCT/CN2020/136452). As expected, the combination of humanized anti-GPC3 antibody with mutant CD3 binders CD31a3b (EC50, H1L1: 0.01 nM and H1L3: 0.004 nM) and CD3 1a3b2b1 (EC50, H1L1: 0.0076 nM and H1L3: 0.00086 nM) that have lower binding affinity to CD3 reduced cytotoxic activity as compared to pairing with wild-type CD3 binder (EC50, H1L1: 0.00036 nM and H1L3: 0.00013 nM) in coculture of activated PBMCs (
Example 8. In Vivo Efficacy Study of Humanized Anti-GPC3/CD3 Bispecific Antibodies
(81) We evaluated the effect of humanized anti-GPC3/CD3 bispecific antibodies on tumor development in an in vivo xenograft model. NSG mice were injected subcutaneously on the right flank at day 0 with HepG2 cells followed by healthy human donor PBMC administration intravenously once tumor growth reaches 70-100 mm.sup.3. Vehicle (PBS) or treatment antibodies (i.e. humanized anti-GPC3/CD3 bispecific antibodies) were administered intravenously twice a week for the next 14 days starting from the time of PBMC administration. In order to measure anti-tumor response, tumor growth was measured twice weekly with a caliper and calculated. As shown in
(82) Tumor was collected at study end point and investigated for GPC3 expression in tumor cells. Interestingly, 45% of tumor cells were GPC3-positive in the PBS treatment group compared to only 6.43%, 2.13% and 2.5% GPC3-positive cells observed in the H1L1-CD3 1a3b, H1L3-CD3 1a3b and H1L3-CD3 groups respectively (
Example 9. Studies on an Anti-GPC3 Chimeric Antigen Receptor
9.1 Preparation of Chimeric Antigen Receptor Gene Fragments
(83) The present application designs fusion gene fragments of the anti-GPC3 chimeric antigen receptor through gene synthesis technology in the order of the following coding genes: CD8 signal peptide coding gene, anti-GPC3 scFv VH-linker-anti-GPC3 scFv VL coding genes, CD8 hinge region coding gene, CD8 transmembrane (TM) region coding gene, and 4-1BB and CD3 intracellular signal regions coding genes. The expressed chimeric antigen receptor has the amino acid structure of scFv VH-linker-scFv VL-CD8hinge-CD8TM-4-1BB-CD3. The sequence of linker is GGGGSGGGGSGGGGS, the sequence of the CD8 signal peptide is SEQ ID NO: 29, the sequence of the CD8 hinge region is SEQ ID NO: 30, the sequence of the CD8 transmembrane region (CD8TM) is SEQ ID NO: 31, the 4-1BB sequence is SEQ ID NO: 32, and the CD3 sequence is SEQ ID NO: 33.
(84) The pRRLSIN lentiviral vector was synthesized by a whole gene of the pRRLSIN lentiviral vector. The vector contains a human EF1a promoter, and the GFP (green fluorescent protein) sequence was replaced with the EGFRt marker protein sequence to obtain the pRRLSIN-EGFRt vector (see
9.2 Construction of Chimeric Antigen Receptor Lentiviral Expression Vector
(85) The vector system used in this example belongs to the third-generation of self-inactivating lentiviral vector systems. The system consists of three plasmids, packaging plasmids pMDLg-pRRE encoding Gag/Pol protein, pRSV-rev encoding Rev protein; and an envelope plasmid PMD2. G encoding VSV-G protein.
(86) In this example, a lentiviral expression vector that co-expressed the specific CAR (i.e. the anti-GPC3 chimeric antigen receptor) and EGFRt (SEQ ID NO:35) linked by P2A (SEQ ID NO:34) was constructed by linking the target gene obtained in Section 9.1 to the pRRLSIN-EGFRt vector. A recombinant plasmid was formed and named pRRLSIN-GPC3 CAR-P2A-EGFRt (see
(87) The control was named as GC33 Benchmark, which was obtained by fusing GC33 CAR, P2A and EGFRt in tandem. The amino acid sequence of GC33 CAR is set forth in SEQ ID NO: 36.
(88) The target CAR structures obtained in this example are as follows: scFv clone40VH-clone 40VL-CD8hinge-CD8TM-4-1BB-CD3-P2A-EGFRt (G8) scFv VH1-VL1-CD8hinge-CD8TM-4-1BB-CD3-P2A-EGFRt (VH1-VL1) scFv VH1-VL2-CD8hinge-CD8TM-4-1BB-CD3-P2A-EGFRt (VH1-VL2) scFv VH1-VL3-CD8hinge-CD8TM-4-1BB-CD3-P2A-EGFRt (VH1-VL3) scFv VH2-VL1-CD8hinge-CD8TM-4-1BB-CD3-P2A-EGFRt (VH2-VL1) scFv VH2-VL2-CD8hinge-CD8TM-4-1BB-CD3-P2A-EGFRt (VH2-VL2) scFv VH2-VL3-CD8hinge-CD8TM-4-1BB-CD3-P2A-EGFRt (VH2-VL3) scFv VH3-VL1-CD8hinge-CD8TM-4-1BB-CD3-P2A-EGFRt (VH3-VL1) scFv VH3-VL2-CD8hinge-CD8TM-4-1BB-CD3-P2A-EGFRt (VH3-VL2) scFv VH3-VL3-CD8hinge-CD8TM-4-1BB-CD3-P2A-EGFRt (VH3-VL3) GC33 CAR-P2A-EGFRt (GC33 Benchmark)
(89) TABLE-US-00011 TABLE11 RelevantsequencesofCARstructures CD8signal MALPVTALLLPLALLLHAARP(SEQIDNO:29) peptide CD8hinge TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIY (SEQIDNO:30) CD8 IWAPLAGTCGVLLLSLVITLYC(SEQIDNO:31) transmembrane region (CD8TM) 4-1BB KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL(SEQ IDNO:32) CD3 RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPE MGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDG LYQGLSTATKDTYDALHMQALPPR(SEQIDNO:33) P2A GSGATNFSLLKQAGDVEENPGP(SEQIDNO:34) EGFRt LLLVTSLLLCELPHPAFLLIPACGADSYEMEEDGVRKCKKCEGPCR KVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFT HTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRT KQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINW KKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDC VSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNIT CTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAG HVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVV ALGIGLFMRRRHIV(SEQIDNO:35) GC33CAR DVVMTQSPLSLPVTPGEPASISCRSSQSLVHSNANTYLHWYLQKPG QSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC SQNTHVPPTFGQGTKLEIKRGGGGSGGGGSGGGGSQVQLVQSGAE VKKPGASVKVSCKASGYTFTDYEMHWVRQAPGQGLEWMGALDP KTGDTAYSQKFKGRVTLTADESTSTAYMELSSLRSEDTAVYYCTRF YSYTYWGQGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAA GGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCRSKRSRLL HSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSKRGRKKLLYIFK QPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQG QNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYN ELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDAL HMQALPPR(SEQIDNO:36)
9.3 Preparation of Chimeric Antigen Receptor Lentivirus
(90) The pRRLSIN-GPC3 CAR-P2A-EGFRt expression plasmid and pMDLg-pRRE, pRSV-rev and pMD2. G helper plasmids were extracted and mixed with the transfection reagent polyethyleneimine (PEI) in a certain ratio to co-transfect 293T cells. The main steps are as follows:
(91) (1) 293T cells (ATCC CRL-3216) cultured to 5-8 generations were inoculated at a cell density of 710.sup.6 in a 75 cm.sup.3 cell culture flask with DMEM medium (purchased from GIBCO) containing 10% FBS (purchased from GIBCO). The cells were mixed well and put in a CO.sub.2 incubator for 24 hours to prepare for transfection. The culture conditions were 37 C. and 5% CO.sub.2. The next day, cell confluence of about 70-80% was observed, and 293T cells were ready for transfection.
(92) (2) After 24 h, the target expression plasmid pRRLSIN-GPC3 CAR-P2A-EGFRt was mixed with pMDLg-pRRE, pRSV-rev and pMD2. G auxiliary plasmid according to a ratio of 4:3:2:2, and diluted with an Opti-MEM medium (purchased from GIBCO) to obtain a solution A. PEI dilution solution was prepared according to a ratio of total plasmid: PEI=3:1, and diluted with the Opti-MEM medium to obtain a solution B. The liquid A and liquid B were mixed and incubated at room temperature for 15 minutes so as to obtain plasmid-PEI mixture.
(93) (3) The plated 293T cells were taken out, and the plasmid-PEI mixture was slowly added to the cell supernatant medium. The resulting mixture was shaken gently and put in a CO.sub.2 incubator for 4-6 hours. The culture conditions were 37 C. and 5% CO.sub.2. After culture, the medium was replaced with a fresh DMEM medium containing 10% FBS.
(94) (4) After 48 h and 96 h post-transfection, the cell culture supernatant containing the virus was collected and centrifuged at 3000 rpm for 5 min at 4 C. After filtering the supernatant through a 0.45 m filter, it was mixed with PEG8000/NaCl at a ratio of 4:1. After standing at 4 C. for 2 to 3 hours, it was centrifuged at high speed for 30 minutes. The supernatant was discarded and the precipitate was resuspended with precooled T cell medium X-VIVO 15 (Lonza, 04-418Q) or PBS to give a virus concentrate which was stored at 80 C. for later use.
9.4 Lentivirus Titer Detection
(95) In this example, a cell infection method was used to determine the biological activity titer of the lentivirus. The 293T cells were used for lentivirus activity assay, and 110.sup.5 cells were inoculated to each well of a 24-well culture plate. 1 mL of fresh DMEM medium containing 10% FBS was added to each well. The mixture was diluted to a final concentration of 6 g/mL with transfection additive Polybrene. The lentivirus concentrate was serially diluted by 3 to the 5th concentration, added at 1 L/well in duplicate (two repeats for each lentivirus concentrate), and mixed well. The cells were incubated in a CO.sub.2 incubator at 37 C./5% CO.sub.2 for 24 h. After 24 h, the cells were digested, and the positive rate of protein expression of CAR or EGFRt was detected by a flow cytometer using an anti-human IgG(Fab).sub.2 (Jackson ImmunoResearch, 109-065-006) or anti-human EGFRt (Biolegend, 352904) flow dye. The titer was calculated by the following formula: Lentiviral activity titer (TU/mL)=positive ratedilution factor10010.sup.5. The activity titer of lentiviral concentrates of the above CARs (G8, VH1-VL1, VH1-VL2, VH1-VL3, VH2-VL1, VH2-VL2, VH2-VL3, VH3-VL1, VH3-VL2, VH3-VL3, GC33 Benchmark) packaged by PEI transfection method were greater than 110.sup.8 TU/mL (
9.5 Preparation of T lymphocytes
(96) Peripheral blood mononuclear cells (PBMCs) purchased from AllCells were marked with microbeads through a CD3 MicroBeads human-lyophilized Kit (purchased from Miltenyi Biotech). CD3+ T lymphocytes with high purity were selected, with a proportion of CD3 positive T cells over 95%. The purified T cells were activated and proliferated using a human CD3/CD28 T cell activator (Dynabeads Human T-Activator CD3/CD28, Thermo Fisher, 11132D).
9.6 Lentivirus Transduces T lymphocytes
(97) CAR-T cells were obtained by transducing T cells with the lentivirus prepared in Section 9.3. T lymphocytes in Section 9.5 were stimulated for 24-48 hours, then microscopic examination was performed to observe whether T lymphocytes were activated. After activation, the T lymphocytes became larger in volume and elongated or irregular in shape. The activated T lymphocytes were collected, centrifuged and resuspended in a T cell medium X-VIVO 15 (Lonza, 04-418Q) with a final concentration of 10 ng/mL IL-7 and 5 ng/mL IL-15. The final volume was 1 mL, and added into a 12-well culture plate. The lentivirus was diluted to MOI=5 with the same medium and mixed with 110.sup.6 activated T lymphocytes for infection. The cells and the lentivirus mixture were mixed thoroughly and added to the 24-well plate, placed in a 37 C., 5% CO.sub.2 incubator and incubated overnight. The next day, the cells were centrifuged again and the medium was refreshed. The cell density was measured every 2 days thereafter, and the cells were further expanded with the cell density controlled at NMT 210.sup.6 cells/mL. After 48-72 h of lentivirus infection, the expression of different chimeric antigen receptors was detected by flow cytometry. With non-transduced T lymphocytes as a negative control, the positive rates of T lymphocytes expressing different chimeric antigen receptors are shown in Table 12.
(98) TABLE-US-00012 TABLE 12 Positive rates of T lymphocytes expressing different chimeric antigen receptors CAR CAR Positive rate G8 18.8% GC33 Benchmark 12.2% VH1-VL1 73.9% VH1-VL2 78.2% VH1-VL3 79.7% VH2-VL1 68.9% VH2-VL2 74.2% VH2-VL3 74.0% VH3-VL1 69.7% VH3-VL2 75.4% VH3-VL3 80.7%
(99) After being infected with lentivirus packaging different chimeric antigen receptors, T lymphocytes were cultured to about 300-fold expansion on the 9th day, indicating that T lymphocytes expressing different chimeric antigen receptors could be expanded in vitro to a certain extent, providing a guarantee that sufficient quantities could be produced for subsequent in vitro functional studies and in vivo drug efficacy studies in animals.
9.7 In Vitro Toxicity Test
(100) Hepatocellular Carcinoma Tumor Cell
(101) Human HCC cell lines (HepG2, SK-HEP-1) were obtained from ATCC. These cell lines were tested and authenticated by DNA profiling for polymorphic short tandem repeat markers. HCC cells were cultured in DMEM supplemented with 10% FBS. All cells were routinely tested for mycoplasma contamination. Surface GPC3 expression on various human HCC cell lines were detected by flow cytometry.
(102) Target Specific Test
(103) In this example, HepG2 cells (purchased from ATCC, HTB-8065) overexpressing GPC3 protein were used as target cells, SK-HEP-1 cells (purchased from ATCC, HTB-52) which do not express GPC3 protein were used as negative target cells and anti-GPC3 CAR-T cells were used as effector cells according to different E:T (effector cell:target cell) ratios (e.g. 3:1,1:1,1:3). The results of in vitro experiments (
(104) The following specific killing detection method was used: an LDH Release Assay Kit (Dojindo, CK12) was used for assay, which is an INT chromogenic reaction catalyzed by diaphorase, and measures the activity of LDH released during cytotoxicity via colorimetry. Damage to the cell membrane structure caused by cell apoptosis or necrosis will lead to release of enzymes in cytoplasm into the cultures, including lactate dehydrogenase (LDH) with relatively stable enzymatic activity. The cytotoxicity can be quantitatively analyzed by activity assay of LDH released from lysed cells into the cultures. LDH release is considered to be an important indicator of cell membrane integrity and is widely used as a cytotoxicity assay.
(105) The following cytokine detection method was used: Human IFN-gamma ELISA kit (R&D Systems, SIF50) was used for measuring cytokines, which is based on the immobilization of an antigen or antibody and enzymatic labeling of the antigen or antibody. The antigen or antibody that binds to the surface of a solid carrier retains immunological activity, while the enzyme labeled antigen or antibody retains both immunological activity and enzymatic activity. During the assay, the test substance (the antigen or antibody) in the sample are bound to the immobilized antibody or antigen. Non-binding substances are removed by washing, and the enzyme-labeled antigen or antibody is added. In this case, the amount of enzyme immobilized is associated with the amount of the test substance in the sample. After a substrate that reacts with the enzyme is added for color development, the content of the test substance in the sample could be judged by the color for qualitative or quantitative analysis.
(106) TABLE-US-00013 TABLE 13 Results of the specific killing ability of different cells on GPC3-positive tumor cells Effector to Target ratio Specific lysis (%) 3:1 1:1 1:3 G8 43.81% 8.66% 2.01% GC33 Benchmark 17.17% 9.78% 4.57% VH1-VL1 70.41% 19.37% 6.99% VH1-VL2 46.19% 8.94% 2.24% VH1-VL3 26.12% 5.58% 1.81% VH2-VL1 56.49% 6.72% 1.73% VH2-VL2 34.24% 6.40% 1.85% VH2-VL3 50.82% 8.85% 2.33% VH3-VL1 27.05% 4.89% 1.50% VH3-VL2 48.14% 8.80% 1.79% VH3-VL3 47.6% 8.26% 1.81%
(107) TABLE-US-00014 TABLE 14 Results of the specific killing ability of different cells on GPC3-negative tumor cells Effector to Target ratio Specific lysis (%) 3:1 1:1 1:3 G8 0.96% 0.30% 0.24% GC33 Benchmark 3.75% 0.51% 0.31% VH1-VL1 0.07% 0.10% 0.07% VH1-VL2 0.59% 0.07% 0.04% VH1-VL3 0.85% 0.26% 0.36% VH2-VL1 0.39% 0.15% 0.23% VH2-VL2 0.79% 0.33% 0.10%3 VH2-VL3 0.74% 0.18% 0.304% VH3-VL1 1.49% 0.65% 0.36% VH3-VL2 1.01% 0.55% 0.41% VH3-VL3 1.78% 0.60% 0.58%
(108) TABLE-US-00015 TABLE 15 Results of IFN-gamma cytokine released in the supernatant of anti-GPC3 CAR-T cocultured with GPC3-positive tumor cells. IFN-gamma cytokine release Effector to Target ratio (pg/mL) 3:1 1:1 1:3 G8 18804.80 8216.72 2103.28 GC33 Benchmark 15021.38 8180.09 2761.31 VH1-VL1 27612.18 11133.82 4331.44 VH1-VL2 23127.24 5813.45 3021.09 VH1-VL3 23304.62 9417.20 2189.11 VH2-VL1 25867.36 9348.75 3792.64 VH2-VL2 19251.72 7675.05 2386.92 VH2-VL3 20025.15 7904.89 2415.53 VH3-VL1 27571.58 6655.57 1956.82 VH3-VL2 19723.78 5412.93 1689.79 VH3-VL3 20670.78 6543.04 2110.36
(109) TABLE-US-00016 TABLE 16 Results of IFN-gamma cytokine released in the supernatant of anti-GPC3 CAR-T cocultured with GPC3-negative tumor cells. IFN-gamma cytokine release Effector to Target ratio (pg/mL) 3:1 1:1 1:3 G8 59.08 146.29 0 GC33 Benchmark 5.64 60.83 0 VH1-VL1 314.21 0 20.40 VH1-VL2 178.82 16.58 7.31 VH1-VL3 202.70 0 18.30 VH2-VL1 690.03 109.55 27.91 VH2-VL2 320.54 65.98 13.57 VH2-VL3 254.74 80.37 15.97 VH3-VL1 394.62 65.72 13.27 VH3-VL2 195.91 56.94 8.54 VH3-VL3 198.85 75.22 13.27
(110) Based on in vitro cytotoxicity tests, it was shown that T lymphocytes expressing different chimeric antigen receptors can have good killing effects on GPC3-positive tumor cells, which provides a reasonable basis for the study of animal in vivo drug efficacy.
9.8 Animal Model Test
(111) In this example, a pharmacodynamic model of immunodeficient mouse bearing Hepatocellular Carcinoma tumor was established. HepG2 cancer cell line was maintained in vitro as a monolayer culture in EMEM medium supplemented with 10% fetal calf serum in a humidified incubator at 37 C. in an atmosphere with 5% CO.sub.2. The HepG2 tumor cells were routinely sub-cultured by trypsin-EDTA treatment three times a week. The HepG2 tumor cells in an exponential growth phase were harvested and counted for inoculation. 110.sup.7 HepG2 tumor cells were injected into the skin of female NCG mice (purchased from GemPharmatech) based on in vivo studies. Anti-GPC3 CAR-T cells were administered on the 16th day after inoculation (tumor volume was around 80-120 mm.sup.3), the solvent control group (Vehicle) was administered with 0.9% saline, the Mock T (T cells not transfected with plasmid) group was administered with 210.sup.7 cells, anti-GPC3 CAR-T low dose and high-dose groups (positive cells) were administered with 3.0010.sup.6 and 1.0010.sup.7 respectively. The administration volume of solvent control group (Vehicle) was 200 l The administration volume of Mock T and CAR-T groups was 100 L. 6 animals were allocated in each group. Tumor volume was measured twice a week after administration; a tumor growth curve was drawn, TGI and T/C were calculated, and all tumors were photographed at the end of the experiment. Blood was collected before CAR-T administration (day-2), 2, 9 and 28 days after administration, and the copy number (VCN) of CAR in the peripheral blood of mice was detected by qPCR to confirm the expansion of CAR-T cells. The measurement of tumor size is conducted with a caliper and the tumor volume (mm.sup.3) was estimated using the formula: TV=0.5ab.sup.2, where a and b are long and short diameters of a tumor respectively. (1) Anti-tumor effect: The results showed that after 12 days of administration of CAR-T, the efficacy of each drug group was significant. Among them, the tumors of 6 mice in the high dose group of VH1-VL1 (110.sup.7 cells) completely regressed (6/6), 5 of 6 mice in the low dose group of VH1-VL1 (310.sup.6 cells) completely regressed (5/6), and 4 of 6 mice in the GC33 Benchmark group (110.sup.7 cells) completely regressed (4/6) (
Other Embodiments
(112) It is to be understood that while the disclosure has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the disclosure, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.
(113) The various embodiments described above can be combined to provide further embodiments. All of the U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications and non-patent publications referred to in this specification and/or listed in the Application Data Sheet are incorporated herein by reference, in their entirety. Aspects of the embodiments can be modified to employ concepts of the various patents, applications and publications to provide yet further embodiments.
(114) These and other changes can be made to the embodiments in light of the above-detailed description. In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and the claims, but should be construed to include all possible embodiments along with the full scope of equivalents to which such claims are entitled. Accordingly, the claims are not limited by the disclosure.
CITATION LIST
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