Patent Application
20240117035
The present application relates to CLDN18.2 antibodies or antigen binding fragments thereof and related CLDN18.2/CD3 bispecific antibodies, as well as corresponding nucleic acids encoding same, vectors comprising such nucleic acids, host cells transfected with such nucleic acids or vectors. The present application further relates to isolated CLDN18.2 antibodies or antigen binding fragments thereof and related CLDN18.2/CD3 bispecific antibodies, pharmaceutical compositions comprising such antibodies, antigen binding fragments, bispecific antibodies, nucleic acids, vectors, or host cells and optionally a pharmaceutically acceptable carrier, and methods of treating cancer in a subject in need thereof by administering such pharmaceutical compositions. The cancers treated in accordance with the present application include CLDN18.2-positive cancers, such as, inter alia, gastric cancer and pancreatic cancer.
CLDN18.2 protein is a transmembrane protein, which belongs to the Claudins (CLDNs) family. The entire protein is expressed on the cell membrane and is an important structural component of the tight junction of cells. Claudins have 4 transmembrane regions, 2 extracellular loops and 1 intracytoplasmic loop, which participate in the formation of tight junctions between cells. There are two alleles in the first exon of human CLDN18 gene, which can express two different splicing mutants known as CLDN18.1 and CLDN18.2, resulting in a 69 amino acid sequence at the N-terminal including extracellular loop 1. Therefore, there are differences between the two extracellular epitopes. Although most CLDNs are widely expressed, individual members are often highly selectively expressed in specific tissues.
Among them, CLDN18.1 protein is a specific antigen selectively expressed by alveolar epithelial cells, which is only highly expressed in normal alveolar tissues, but not found in other normal tissues, including pancreatic ducts; CLDN18.2 protein is also a highly selective marker protein, but its distribution is completely different than CLDN18.1 protein. The expression of CLDN18.2 protein is highly restricted in normal healthy tissues. It is not expressed in undifferentiated gastric stem cells. It is only expressed in differentiated gastric mucosal membrane epithelial cells, and the expression level is very limited. This is conducive to maintaining the gastric mucosa, whose barrier function can prevent H+ in gastric acid from leaking through paracellular pathways. However, CLDN18.2 protein frequently undergoes abnormal changes during the development of a variety of malignant tumors. For example, when gastric epithelial tissue undergoes malignant transformation, the resultant disorder of cell polarity will cause the CLDN18.2 protein epitope on the cell surface to be exposed. At the same time, CLDN18.2 gene will also be abnormally activated, and highly selectively and stably expressed in specific tumor tissues involved in the proliferation, differentiation, and migration of tumor cells.
In recent years, CLDN18.2 has become a promising target for tumor therapy. So far, nearly 20 innovative pharmaceutical companies have deployed CLDN18.2-targeted drug development. Most of these research projects are single-target antibody drugs. Among them, CLDN18.2 specific antibody Claudiximab (Zolbetuximab/IMAB362) has achieved remarkable success in clinical trials. A phase II study from Europe showed that compared with chemotherapy alone, the overall survival time of patients with advanced gastric cancer was extended from 8.4 months to 13.2 months after treatment with IMAB362 and standard chemotherapy. In this study, patients with the highest claudin 18.2 levels had a longer median overall survival time (16.7 months). Moreover, CLDN18.2 double-antibody drugs have also ushered in exciting new developments in solid tumor therapy. Recently, a new CLDN18.2/CD3 bispecific antibody, AMG 910, has been approved for Phase I clinical trials for gastric cancer and gastroesophageal junction cancer.
Overall, the evidence from current clinical trials has demonstrated that CLDN18.2 antibodies have high safety, tolerability, and anti-tumor activity as a targeted drug for patients with CLDN18.2-positive advanced gastric cancer.
The present application provides novel CLDN18.2 antibodies or antigen binding fragments thereof and also related bispecific antibodies with particularly advantageous properties such as high producibility, stability, binding affinity, biological activity, specific targeting of CLDN18.2-positive cells, targeting efficiency, remaining tumor cell killing and/or reduced toxicity.
In one aspect, the present application provides a CLDN18.2 antibody or antigen binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises HCDR1 comprising the amino acid sequence represented by SEQ ID NO. 11, HCDR2 comprising the amino acid sequence represented by SEQ ID NO. 12 and HCDR3 comprising the amino acid sequence represented by SEQ ID NO. 13, and/or the light chain variable region comprises LCDR1 comprising the amino acid sequence represented by SEQ ID NO. 14, LCDR2 comprising the amino acid sequence represented by SEQ ID NO. 15 and LCDR3 comprising the amino acid sequence represented by SEQ ID NO. 16.
In another aspect of the present application, the antigen binding fragment is selected from scFv fragment, Fv fragment, F(ab′)2 fragment, Fab′-SH fragment and Fab′ fragment.
In a further aspect, the CLDN18.2 antibody or antigen binding fragment binds to CLDN 18.2, and does not bind to CLDN 18.1.
In a further aspect, the above-mentioned antibody is a monoclonal antibody or a humanized antibody.
In a further aspect, such monoclonal antibody or humanized antibody includes antibodies produced by hybridomas or host cells transformed with an expression vector modified to carry an antibody gene by genetic engineering techniques. In a further embodiment, the monoclonal antibody or humanized antibody may comprise IgG antibodies comprising two heavy chains and two light chains; heavy chains of mouse IgG antibodies are classified into five subclasses according to the constant region of their heavy chains: IgG1, IgG2a, IgG2b, IgG2c and IgG3, and the light chains may be mouse lambda or kappa type; and heavy chains of human IgG antibodies are further classified into four subclasses according to the constant region of their heavy chains: IgG1, IgG2, IgG3, and IgG4, and the light chains may be human lambda or kappa type.
In a further aspect, the disclosure also provides a monoclonal CLDN18.2 antibody including a light chain variable region and a heavy chain variable region, wherein the heavy chain variable region comprises an amino acid sequence with at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence of SEQ ID NO. 1, and/or the light chain variable region comprises an amino acid sequence with at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence of SEQ ID NO. 2.
In a further aspect, the disclosure also provides a monoclonal CLDN18.2 antibody including a light chain variable region and a heavy chain variable region, wherein the heavy chain variable region comprises an amino acid sequence with at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence of SEQ ID NO. 3, and/or the light chain variable region comprises an amino acid sequence with at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence of SEQ ID NO. 4.
In a further aspect, the disclosure also provides a monoclonal CLDN18.2 antibody including a light chain variable region and a heavy chain variable region, wherein the heavy chain variable region comprises an amino acid sequence with at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence of SEQ ID NO. 5, and/or the light chain variable region comprises an amino acid sequence with at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence of SEQ ID NO. 6.
In a further aspect, the disclosure also provides a monoclonal CLDN18.2 antibody including a light chain variable region and a heavy chain variable region, wherein the heavy chain variable region comprises an amino acid sequence with at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence of SEQ ID NO. 7, and/or the light chain variable region comprises an amino acid sequence with at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence of SEQ ID NO. 8.
In a further aspect, the disclosure also provides a monoclonal CLDN18.2 antibody including a light chain variable region and a heavy chain variable region, wherein the heavy chain variable region comprises an amino acid sequence with at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence of SEQ ID NO. 9, and/or the light chain variable region comprises an amino acid sequence with at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence of SEQ ID NO. 10.
In a further aspect, the antibody or antigen-binding fragment thereof is a humanized antibody or antigen-binding fragment thereof, wherein the humanized CLDN18.2 antibody includes a light chain variable region and a heavy chain variable region, wherein the heavy chain variable region comprises an amino acid sequence with at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence of any one of SEQ ID NOs. 17-21, and/or the light chain variable region comprises an amino acid sequence with at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence of any one of SEQ ID NOs. 22-24.
In a further aspect, the antibody or antigen-binding fragment thereof is a humanized antibody or antigen-binding fragment thereof, the humanized CLDN18.2 antibody includes a light chain variable region and a heavy chain variable region, wherein the heavy chain variable region comprises an amino acid sequence with at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence of SEQ ID NO. 17, and the light chain variable region comprises an amino acid sequence with at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence of SEQ ID NO. 22.
In a further embodiment, the antibody comprises a light chain constant domain, and optionally a heavy chain constant domain.
Optionally, the heavy chain constant domain is a human IgG1 heavy chain constant domain, or the light chain constant domain is a human kappa light chain constant domain. In a further embodiment, the heavy chain constant domain may contain one or more mutations, for example, mutations L234A, L235A and P329A, that reduce or eliminate an effect of ADCC and/or CDC.
In a specific aspect, the present application provides a CD3 antigen binding domain that specifically binds to CD3, wherein the antigen binding fragment comprises a heavy chain variable region and a light chain variable region, wherein
In a specific aspect, the present application provides a CD3 antigen binding domain that specifically binds to CD3, wherein the antigen binding fragment comprises a heavy chain variable region (VH domain) and a light chain variable region (VL domain) wherein:
In a further aspect, the present application provides a bispecific antibody, comprising a first antibody or antigen binding fragment thereof that specifically binds to CLDN18.2 or a naturally occurring variant thereof and a second antigen binding domain that specifically binds to CD3 or a naturally occurring variant thereof.
In a further aspect, in the above-mentioned bispecific antibody, the first antibody or antigen binding fragment thereof is the above-mentioned CLDN18.2 antibody or antigen binding fragment thereof, and the second antigen binding domain is the above-mentioned CD3 antigen binding domain.
In a further aspect, in the above-mentioned bispecific antibody, the first antibody or antigen binding fragment thereof comprises two identical heavy chain and two identical light chains, and the second antigen binding domain comprises two identical single chain antibody fragments (scFv).
In a further aspect, in the above-mentioned bispecific antibody, each of the light chains of the first antibody or antigen binding fragment is fused to each said single chain antibody fragment (scFv) of the second antigen binding domain.
In a further aspect, in the above-mentioned bispecific antibody, the C-terminal of the constant region of each said light chain of the first antibody or antigen binding fragment is fused to the N-terminal of the heavy chain variable region of each said single chain antibody fragment (scFv) of the second antigen binding domain, directly or via a peptide linker.
In a further aspect, the above-mentioned bispecific antibody comprises a monoclonal antibody that is an immunoglobulin that binds to CLDN18.2, said immunoglobulin comprising two identical heavy chains and two identical light chains, wherein said light chains are a first light chain and a second light chain, wherein the first light chain is fused to a first single chain variable fragment (scFv), via a peptide linker, to create a first light chain fusion polypeptide, and wherein the second light chain is fused to a second scFv, via a peptide linker, to create a second light chain fusion polypeptide, wherein the first and second scFv (i) are identical, and (ii) bind to CD3, and wherein the first and second light chain fusion polypeptides are identical.
In one specific aspect, in the above-mentioned bispecific antibody, the C-terminal of the constant region of the first light chain is fused to the N-terminal of the heavy chain variable region of the first scFv directly or via a peptide linker, and the C-terminal of the constant region of the second light chain is fused to the N-terminal of the heavy chain variable region of the second scFv directly or via a peptide linker.
In one specific aspect, in the above-mentioned bispecific antibody, each of the first and second scFv comprises the above mentioned CD3 antigen binding domain.
In a specific aspect, the above-mentioned bispecific antibody provided herein binds to both CLDN18.2 and CD3.
In a specific aspect, the sequence of the above-mentioned peptide linker is shown in SEQ ID NO. 41 or SEQ ID NO. 42.
In a further aspect, the present application provides a bispecific antibody, comprising a first antibody or antigen binding fragment thereof that specifically binds to CLDN18.2 or a naturally occurring variant thereof and a second antigen binding domain that specifically binds to CD3 or a naturally occurring variant thereof, wherein a first antibody or antigen binding fragment thereof includes a light chain variable region and a heavy chain variable region, wherein the heavy chain variable region comprises HCDR1 comprising the amino acid sequence represented by SEQ ID NO. 11, HCDR2 comprising the amino acid sequence represented by SEQ ID NO. 12 and HCDR3 comprising the amino acid sequence represented by SEQ ID NO. 13, and the light chain variable region comprises LCDR1 comprising the amino acid sequence represented by SEQ ID NO. 14, LCDR2 comprising the amino acid sequence represented by SEQ ID NO. 15 and LCDR3 comprising the amino acid sequence represented by SEQ ID NO. 16; and the second antigen binding domain comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1 comprising the amino acid sequence represented by SEQ ID NO. 31, HCDR2 comprising the amino acid sequence represented by SEQ ID NO. 32 and HCDR3 comprising the amino acid sequence represented by SEQ ID NO. 33, and the light chain variable region comprises LCDR1 comprising the amino acid sequence represented by SEQ ID NO. 34, LCDR2 comprising the amino acid sequence represented by SEQ ID NO. 35 and LCDR3 comprising the amino acid sequence represented by SEQ ID NO. 36.
In a further aspect, the present application provides a bispecific antibody, comprising a first antibody or antigen binding fragment thereof that specifically binds to CLDN18.2 or a naturally occurring variant thereof and a second antigen binding domain that specifically binds to CD3 or a naturally occurring variant thereof, wherein a first antibody or antigen binding fragment thereof includes a light chain variable region and a heavy chain variable region, wherein the heavy chain variable region comprises HCDR1 comprising the amino acid sequence represented by SEQ ID NO. 11, HCDR2 comprising the amino acid sequence represented by SEQ ID NO. 12 and HCDR3 comprising the amino acid sequence represented by SEQ ID NO. 13, and the light chain variable region comprises LCDR1 comprising the amino acid sequence represented by SEQ ID NO. 14, LCDR2 comprising the amino acid sequence represented by SEQ ID NO. 15 and LCDR3 comprising the amino acid sequence represented by SEQ ID NO. 16; and the second antigen binding domain comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1 comprising the amino acid sequence represented by SEQ ID NO. 37, HCDR2 comprising the amino acid sequence represented by SEQ ID NO. 38 and HCDR3 comprising the amino acid sequence represented by SEQ ID NO. 39, and the light chain variable region comprises LCDR1 comprising the amino acid sequence represented by SEQ ID NO. 34, LCDR2 comprising the amino acid sequence represented by SEQ ID NO. 35 and LCDR3 comprising the amino acid sequence represented by SEQ ID NO. 36.
In a further aspect, the present application provides a bispecific antibody, comprising a first antibody or antigen binding fragment thereof that specifically binds to CLDN18.2 or a naturally occurring variant thereof and a second antigen binding domain that specifically binds to CD3 or a naturally occurring variant thereof, wherein a first antibody or antigen binding fragment thereof includes a light chain variable region and a heavy chain variable region, wherein the heavy chain variable region comprises HCDR1 comprising the amino acid sequence represented by SEQ ID NO. 11, HCDR2 comprising the amino acid sequence represented by SEQ ID NO. 12 and HCDR3 comprising the amino acid sequence represented by SEQ ID NO. 13, and the light chain variable region comprises LCDR1 comprising the amino acid sequence represented by SEQ ID NO. 14, LCDR2 comprising the amino acid sequence represented by SEQ ID NO. 15 and LCDR3 comprising the amino acid sequence represented by SEQ ID NO. 16; and the second antigen binding domain comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1 comprising the amino acid sequence represented by SEQ ID NO. 37, HCDR2 comprising the amino acid sequence represented by SEQ ID NO. 40 and HCDR3 comprising the amino acid sequence represented by SEQ ID NO. 39, and the light chain variable region comprises LCDR1 comprising the amino acid sequence represented by SEQ ID NO. 34, LCDR2 comprising the amino acid sequence represented by SEQ ID NO. 35 and LCDR3 comprising the amino acid sequence represented by SEQ ID NO. 36.
In a further aspect, the present application provides a bispecific antibody, comprising a first antibody or antigen binding fragment thereof that specifically binds to CLDN18.2 or a naturally occurring variant thereof and a second antigen binding domain that specifically binds to CD3 or a naturally occurring variant thereof, wherein a first antibody or antigen binding fragment thereof includes a light chain variable region and a heavy chain variable region, wherein the heavy chain variable region comprises an amino acid sequence with at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence of SEQ ID NO. 17, and the light chain variable region comprises an amino acid sequence with at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence of SEQ ID NO. 22; and the second antigen binding domain comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises an amino acid sequence with at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence of SEQ ID NO. 25, and the light chain variable region comprises an amino acid sequence with at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence of SEQ ID NO. 26.
In a further aspect, the present application provides a bispecific antibody, comprising a first antibody or antigen binding fragment thereof that specifically binds to CLDN18.2 or a naturally occurring variant thereof and a second antigen binding domain that specifically binds to CD3 or a naturally occurring variant thereof, wherein a first antibody or antigen binding fragment thereof includes a light chain variable region and a heavy chain variable region, wherein the heavy chain variable region comprises an amino acid sequence with at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence of SEQ ID NO. 17, and the light chain variable region comprises an amino acid sequence with at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence of SEQ ID NO. 22; and the second antigen binding domain comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises an amino acid sequence with at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence of SEQ ID NO. 27, and the light chain variable region comprises an amino acid sequence with at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence of SEQ ID NO. 26.
In a further aspect, the present application provides a bispecific antibody, comprising a first antibody or antigen binding fragment thereof that specifically binds to CLDN18.2 or a naturally occurring variant thereof and a second antigen binding domain that specifically binds to CD3 or a naturally occurring variant thereof, wherein a first antibody or antigen binding fragment thereof includes a light chain variable region and a heavy chain variable region, wherein the heavy chain variable region comprises an amino acid sequence with at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence of SEQ ID NO. 17, and the light chain variable region comprises an amino acid sequence with at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence of SEQ ID NO. 22; and the second antigen binding domain comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises an amino acid sequence with at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence of SEQ ID NO. 28, and the light chain variable region comprises an amino acid sequence with at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence of SEQ ID NO. 26.
In a further aspect, the above-mentioned bispecific antibodies provided herein have advantageous properties such as high producibility, stability, binding affinity, biological activity, specific targeting of certain T cells, targeting efficiency, remaining tumor cell killing and reduced toxicity.
In a further aspect, the above-mentioned bispecific antibody provided here shows reduced binding affinity with CD3 compared to wild-type CD3 binders.
In a further aspect, the above-mentioned bispecific antibodies provided herein show reduced TCR signaling strength.
In a further aspect, the above-mentioned bispecific antibodies provided herein show reduced cytokine release mediated by mutant CD3 binder variants.
In a further aspect, the present application also provides an isolated nucleic acid comprising nucleic acid sequences encoding the above-described CLDN18.2 antibody or antigen binding fragment thereof or the above-mentioned bispecific antibody.
In a further aspect, the present application also provides a vector comprising the nucleic acid encoding the above-mentioned CLDN18.2 antibody or antigen binding fragments thereof or the above-mentioned bispecific antibody.
In a further aspect, the disclosure also provides an isolated host cell comprising the above-mentioned vector or the above-mentioned isolated nucleic acid.
The appropriate host cells are transformed with the above-mentioned vector, and the above-mentioned host cells expressing the above-mentioned CLDN18.2 antibody or antigen binding fragments thereof or the above-mentioned bispecific antibody are obtained.
In a further aspect, the disclosure also provides various known host cell/expression vector combinations for antibody preparation by introducing isolated antibody genes into appropriate hosts. Appropriate eukaryotic cells used as host cells include animal cells, plant cells, and fungal cells. Specifically, the animal cells include, for example, the following cells: (1) mammalian cells: CHO, COS, myeloma, baby hamster kidney (BHK), HeLa, Vero, or such; (2) amphibian cells: Xenopus oocytes, or such; and (3) insect cells: sf9, sf21, Tn5, or such.
In a further aspect, the disclosure also provides a method of producing the above-mentioned antibody or antigen binding fragment thereof or the above-mentioned bispecific antibody, comprising culturing the above-mentioned host cell so that the antibody or antigen binding fragment thereof or the bispecific antibody is produced. In a further aspect, the method further comprises recovering the antibody or antigen binding fragment thereof or the bispecific antibody produced by the host cell.
In a further aspect, the disclosure also provides the isolated antibody or antigen binding fragment thereof or the bispecific antibody produced by the above-mentioned method.
In a further aspect, the disclosure also provides a pharmaceutical composition comprising the above-mentioned CLDN18.2 antibody or antigen binding fragment thereof or the above-mentioned bispecific antibody, nucleic acid, vector, the host cell and optionally a pharmaceutically acceptable carrier.
In a further aspect, the disclosure also provides a method of treating cancer in a subject in need thereof, comprising administering to the subject the above-mentioned pharmaceutical composition comprising the above-mentioned CLDN18.2 antibody or antigen binding fragment thereof or the above-mentioned bispecific antibody, nucleic acid, vector, the host cell and optionally a pharmaceutically acceptable carrier.
In a further aspect, the above-mentioned cancer is a CLDN18.2-positive cancer.
In a further aspect, the cancer is hematological cancer or solid cancer.
In a further aspect, the CLDN18.2-positive cancer includes but is not limited to gastric cancer or pancreatic cancer.
The technical solutions of the present application have one or more of the following advantages:
The novel features of the present 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.
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.
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.
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.
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 are known in the art. 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.
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 α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM), some of which may be further divided into subtypes, e.g., γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), γ4 (IgG4), α1 (IgA1) and α2 (IgA2). The light chain of an antibody may be assigned to one of two types, called kappa (κ) and lambda (λ), 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.
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.
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 art, 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 μM, <100 nM, <10 nM, <1 nM, <0.1 nM, <0.01 nM, or <0.001 nM (e.g., 10-7 M or less, e.g., from 10-7 M to 10-13 M, e.g., from 10-9 M to 10-13 M).
“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 (Kd), which is the ratio of dissociation and association rate constants (koff and kon, 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 Kd of 10−9 M or less and even more particularly 10−10 M or less for a target antigen. The term “low affinity” of an antibody refers to an antibody having a Kd of 10−8 or higher.
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.
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 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.
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 L1, 50-55 of L2, 89-96 of L3, 31-35B of H1, 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.
By “fused to” or “connected to”, it 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.
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.
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.
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.
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 present application are used to delay development of a disease or to slow the progression of a disease.
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 Ewing's sarcoma, including refractory versions of any of the above cancers, or a combination of one or more of the above cancers.
For purposes of interpreting the antibodies used in the following examples, the following definitions are provided.
SSQSLLNSGNQKNYLTWYQQKPGQ
AYTYPLTFGAGTKLEIK
GNGDTKYNGNFKGRVTITRDTSASTAYMELSSLRSEDTAVYYCARYYRGNCFDY
GNGDTKYNGNFKGRVTITADTSASTAYMELSSLRSEDTAVYFCARYYRGNCFD
YWGQGTLVTVSS(SEQ ID NO. 18)
GNGDTKYNGNFKGRVTITADTSASTAYMELSSLRSEDTAVYFCARYYRGNCFD
YWGQGTLVTVSS(SEQ ID NO. 19)
GNGDTKYNGNFKGRVTLTADTSASTAYMELSSLRSEDTAVYFCARYYRGNCFDY
GNGDTKYNGNEKGRATLTADTSASTAYMELSSLRSEDTA VYFCARYYRGNCF
ASTRDSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNAYTYPLTFGGGTKVEI
ASTRDSGVPDRFSGSGSGTDFTLTISSVQAEDVAVYYCQNAYTYPLTFGGGTKVEI
WASTRDSGVPDRFSGSGSGTDFTLTISSVQAEDVAVYYCQNAYTYPLTFGGGTKV
TGAVTSGYYPNWVQQKPGQAPRGL
WVFGGGTKLTVL
TGAVTSGYYPNWVQQKPGQAPRGL
WVFGGGTKLTVL
TGAVTSGYYPNWVQQKPGQAPRGL
WVFGGGTKLTVL
1. Lead Murine Anti-Human CLDN18.2 Antibodies and Humanized Anti-CLDN18.2 Antibodies
We selected three lead murine anti-human CLDN18.2 antibodies (m-anti-human CLDN18.2-6 (also referred to as m-6), m-anti-human CLDN18.2-12 (also referred to as m-12), and m-anti-human CLDN18.2-13 (also referred to as m-13), and two humanized CLDN18.2 antibodies hCLDN18.2 808 and hCLDN18.2 841. VH and VL sequences thereof are listed in Table 1.
2. The Kinetics of CLDN18.2 Monoclonal Antibody m-13 Binding to hCLDN18.2 Protein were Measured Using a BIAcore 8K Instrument Based on Surface Plasmon Resonance (SRP) Technology.
VH and VL sequences of m-13 are shown in Table 1, the heavy chain constant region of m-13 is the constant region of mouse IgG2a heavy chain, and the light chain of m-13 is a mouse kappa light chain. The sequences thereof are listed in Table 4.
For kinetic measurements, hCLDN18.2 protein (Genscript) was serially diluted with HBS-EP+1× (cytiva, BR-1006-69) buffer, starting at 50 nM, with 4 concentration gradients of 2-fold dilution and set 0 concentration. Startup 3 times. m-13 antibody: 2 μg/mL, injection time 100 s, flow rate 10 μL/min, captured with ProA chip (cytiva, 29127556); antigen protein hCLDN18.2(Genscript): binding for 120 s, flow rate 30 μL/min, dissociation for 600 s; regeneration: regeneration with MgCl2 buffer for 30 s, flow rate 30 μL/min.
The binding constant (ka) and dissociation constant (kd) were calculated using the 1:1 binding model (BIAcore Evaluation Software version 3.2), and the Equilibrium Dissociation Constant (KD) was calculated as the ratio of kd/ka. The results can be seen in
3. Confirmation of Binding Affinity of CLDN18.2 Monoclonal Antibody m-13 to CLDN18.2 Overexpressing HEK293 Cells and SNU601 Cells
3.1 We First Compared Binding Affinity of the CLDN18.2 Monoclonal Antibodies to Human CLDN18.2-Overexpressing HEK293 Cells.
To determine binding of CLDN18.2 monoclonal antibody on the cell surface; fifty thousand hCLDN18.2-expressing HEK293 cells (KYinno) per well were placed in a 96-well V-bottom plate (Corning) and incubated with test monoclonal antibody m-13 on ice for 30 minutes. HEK293 cells were washed with FACS buffer (PBS, 2% FBS, 1 mM EDTA) and stained with goat anti-human-IgG-PE Fab (Jackson Immuno Research) on ice for 30 minutes. HEK293 cells were washed twice with FACS buffer and DAPI was added to the final suspension, and the apparent binding activity was assessed by flow cytometry and the results are shown in
3.2 We Further Compared Binding Affinity of the CLDN18.2 Monoclonal Antibodies to Human SNU601 Cells.
To determine binding of CLDN18.2 monoclonal antibody on the cell surface; fifty thousand SNU601 cells (Cobioer) per well were placed in a 96-well V-bottom plate (Corning) and incubated with test monoclonal antibody m-13 on ice for 30 minutes. SNU601 cells were washed with FACS buffer (PBS, 2% FBS, 1 mM EDTA) and stained with goat anti-human-IgG-PE Fab (Jackson Immuno Research) on ice for 30 minutes. SNU601 cells were washed twice with FACS buffer and DAPI was added to the final suspension, and the apparent binding activity was assessed by flow cytometry and the results are shown in
1. Generation of CLDN18.2/CD3 Bispecific Antibodies and Evaluation of the Bispecific Antibodies and Lead CLDN18.2/CD3 Bispecific Antibodies Candidate Selection
1.1. Generation of CLDN18.2/CD3 Bispecific Antibodies
The above two lead humanized CLDN18.2 antibodies (hCLDN18.2 808 and hCLDN18.2 841) and three lead murine anti-human CLDN18.2 antibodies (m-anti-human CLDN18.2-6 (also referred to as m-6), m-anti-human CLDN18.2-12 (also referred to as m-12), and m-anti-human CLDN18.2-13 (also referred to as m-13) were used to develop a new CLDN18.2/CD3 bispecific antibody (bispecific antibody against human CLDN18.2 and CD3) for CLDN18.2 positive tumor therapy. Humanized anti-CD3 antibody: SP34(i.e. CD3-p or CD3 Parental), CD3-v1, or CD3-v2 were used as one arm to construct CLDN18.2/CD3 bispecific antibody in a butterfly format (
In this butterfly format, Fab of anti-CLDN18.2 antibody is used to bind CLDN18.2 expressed on tumor cells and two anti-CD3 scFv-arms fused to the C-terminus of the light chains of anti-CLDN18.2 is used to bind to CD3 on T cells. This format is bivalent for CLDN18.2 and functionally monovalent for CD3. Specifically, for the bispecific antibodies, anti-CLDN18.2 clone variable regions (VH and VL) were fused to the constant regions of human IgG1 heavy chain and human kappa light chain respectively; the sequences of the constant regions are listed in Table 4. The anti-CD3 single-chain variable fragment (scFv) is fused to the C terminus of the light chains of anti-CLDN18.2. To avoid Ig Fc mediated interactions, mutations L234A, L235A and P329A were introduced in the human IgG1 Fc region to eliminate effector functions (e.g. ADCC/CDC function). CLDN18.2/CD3 bispecific antibody with conventional Fc (e.g. Fc of IgG1, IgG2, IgG3 or IgG4) can also mediate killing effect.
1.2. Evaluation of Binding Affinity of the CLDN18.2/CD3 Bispecific Antibodies (i.e. CLDN18.2/CD3 bsAbs) to Human CLDN18.2-Overexpressing HEK293 Cells
We first compared binding affinity of the CLDN18.2/CD3 bsAbs to human CLDN18.2-overexpressing HEK293 cells.
To determine binding of CLND18.2/CD3 bispecific antibodies on the cell surface; twenty thousand hCLND18.2-overexpressing HEK293 cells(ATCC, CRL-3216) per well were placed in a 96-well V-bottom plate (Corning) and incubated with test bsAbs (h808/CD3-p, h841/CD3-p, m-6/CD3-p, m-13/CD3-p, or AMG-910) on ice for 30 minutes. HEK293 cells were washed with FACS buffer (PBS, 2% FBS, 1 mM EDTA) and stained with goat anti-human-IgG-PE Fab (Jackson Immuno Research) on ice for 30 minutes. Cells were washed twice with FACS buffer and DAPI was added to the final suspension. The apparent binding activity was assessed by flow cytometry and the results are shown in
1.3. Evaluation of Binding Affinity of the CLDN18.2/CD3 Bispecific Antibodies to CD3+ Jurkat Cells
To compare CD3 binding affinity of Amgen CLDN18.2/CD3(AMG-910) bsAb with CLDN18.2/CD3 bsAbs having mutant anti-CD3 arm (h841/CD3-v1 and h841/CD3-v2) or having parental anti-CD3 arm (h841/CD3-p), CD3+ Jurkat cells(ATCC, Clone E6-1) were stained with h841/CD3-p, h841/CD3-v1 as well as h841/CD3-v2 with both CD3-v1 and CD3-v2 having reduced CD3 binding affinity. As shown in
1.4. Evaluation of TCR-NFAT Activity Mediated by CLDN18.2/CD3 bsAb in NFAT-Reporter Jurkat Cells
We further verified TCR-NFAT activity mediated by CLDN18.2/CD3 bsAb in NFAT-reporter Jurkat cells (InvivoGen, jktl-nfat). NFAT activity mediated by AMG-910 bsAb and h841/CD3-p was significantly lower than NFAT activity mediated by h808/CD3-p, m-6/CD3-p, and m-13/CD3-p (
1.5. Evaluation of T Cells Killing Effect Mediated by CLDN18.2/CD3 bsAb in Three Tumor Cell Lines with Various Expression Levels of hCLDN18.2
In selecting a lead candidate antibody, one of the most important functions is whether it can efficiently mediate T cells to kill targets. To evaluate this, we chose three tumor cell lines with various expression levels of hCLDN18.2 as targets. Freshly isolated human PBMCs (STEMCELL Technologies) were cocultured with the target cells in serial dilution of the tested CLDN18.2/CD3 bsAbs at E/T ratio of 20:1 for 48 hours. Killing activity to the target cells was determined by luminescence units as these target cells were transfected with luciferase. As shown in
1.6. Evaluation of Cytokines Release Mediated by CLDN18.2/CD3 bsAb
T-cell engager antibody can mediate direct cell-cell contacts between CTLs and target cells, which results in killing target cells. On the other hand, activated T cells can simultaneously secrete cytokines, such as IFN-α and TNF-γ as long as TCR stimulation continues by antibody engagement. These cytokines affect the target cell or cell distal to the effector T cells. TNF-α engages its receptor on the target cell and triggers target-cell apoptosis, and IFN-γ increases Fas-mediated target-cell lysis. However, although cytokines are necessary for the functioning of T cells, high cytokine release levels mediated by T cell engager antibody would induce a potential unfavorable cytokine release syndrome (CRS). Therefore, proper levels of cytokine release mediated by CLDN18.2/CD3 bispecific antibodies would need to be taken into consideration for evaluation of a lead candidate. For this reason, a parallel study was designed to investigate cytokine release in the same co-culture condition as the killing assay. The target cells and human PBMCs were plated in 96-well microplates at an E:T ratio of 20:1 and co-cultured with various concentrations of anti-CLDN18.2/CD3 bsAbs(h841/CD3-p, AMG-910, m-6/CD3-p, m-12/CD3-p, and m-13/CD3-p). After 48 hours of incubation, cell-free supernatants were harvested, and the production of INF-α, TNF-γ, IL-2, and IL-10 secretion was measured by ELISA in the supernatants from the coculture of PBMCs with various CLDN18.2 expressing HEK (i.e. CLDN18.2 HEK), SNU620, and KATO III target cells. In line with cytolytic activity, m-6/CD3-p, m-12/CD3-p, and m-13/CD3-p clones induced higher INF-α (
1.7. Evaluation of Binding Specificity of Anti-CLDN18.2 Antibodies and Lead CLDN18.2/CD3 Bispecific Antibodies Candidate Selection
As CLDN18.1 and CLDN18.2 share highly homologous sequences, the assessment of binding specificity of anti-CLDN18.2 antibodies is critical for the development of therapeutic Claudin18.2 bispecific antibodies. From the killing assays (
As described above, we tested the specificity of h808/CD-3-p, h841/CD3-p, and three murine CLDN18.2/CD3-p BsAbs on human CLDN18.1-overexpressing CHO cells (Creative Biogene, SCS-R00629). In general, after dissociating cells and washing in PBS, 1×105 CLDN18.1 expressing CHO cells were seeded in a 96 well plate. The anti-CLDN18.2/CD3 antibodies prepared in a final concentration of 25 μg/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.
As shown in
To further confirm binding specificity of the murine CLDN18.2 clones m-6/CD3-p, m-12/CD3-p, m-13/CD3-p were tested against CLDN18.2 negative tumor cell lines LoVo (ACTT, CCL-229) and LS-174T (ATCC, LC-188) using flow cytometry assays. As shown in
2. Evaluation of Humanized CLDN18.2-13/CD3 Bispecific Antibodies
2.1 Binding Affinity of CLDN18.2-13/CD3 Bispecific Antibodies
As m-13 and m-6 clones displayed high binding affinity, high specificity, and potent cytotoxicity, m-13 was finally chosen as a lead candidate for humanization and m-6 also was humanized as a back-up clone. The VH and VL sequences of humanized m-6 (i.e., h-6) are shown as SEQ ID Nos. 29-30 listed in Table 2-1, respectively. For m-13, the top 3 humanized variable light chains and 5 variable heavy chains were obtained, which reached high human-ness scores (VL1-90.1%, VL2-89.1%, VL3-88.1%, VH1-86.7%, VH2-82.7%, VH3-81.6%, VH4-80.6%, and VH5-79.6%) (VH and VL sequences are listed in Table 2). To characterize the CLDN18.2-13 antibodies (i.e. CLDN18.2-h-13 antibodies) resulting from those humanized heavy chains (i.e. VH1-VH5) and light chains (i.e. VL1-VL3), each humanized light chain was co-transfected with each humanized CLDN18.2-13 heavy chain in CD3 bispecific format. As a result, 15 humanized CLDN18.2-13/CD3 bispecific antibodies were generated. To determine their apparent binding activity, high CLDN18.2 expressing HEK293 and SNU620 cell lines as targets were incubated in varying concentrations of humanized CLDN18.2/CD3 bispecific antibodies, and with anti-human IgG-PE as a secondary antibody. The cells were then washed twice with FACS buffer. FACS analysis was carried out on a FACS Flow Cytometer. Through flow cytometry assays, we characterized the binding affinity of humanized CLDN18.2-13/CD3 subclones to CLDN18.2 on hCLDN18.2 over-expressing HEK293 cells (high expression of CLDN18.2) and CLDN18.2 natural expressing SNU-620 cells (medium expression of CLDN18.2). We found that all 15 humanized CLDN18.2-13/CD3 candidates had comparable binding affinities as parental m-13 (
2.2 High Specificity of CLDN18.2-13/CD3 Bispecific Antibodies
Although all humanized CLDN18.2-13/CD3 bsAbs retained high binding affinity to CLDN18.2 similar to parental m-13, whether the humanization of m-13 clone would change binding specificity was also very important to determine for the resultant humanized heavy chains and light chains. Therefore, we examined binding specificity of humanized CLDN18.2-13/CD3 subclones by staining human CLDN18.1 overexpressing CHO cells. As shown in
2.3 TCR/CD3 Signaling Strength Mediated by CLDN18.2-13/CD3 Bispecific Antibodies (i.e. h-13/CD3 bsAbs) with CD3-p (i.e. CD3 Parental) Arm.
In addition to binding affinity, TCR/CD3 signaling strength mediated by humanized CLDN18.2-13/CD3 subclones was also validated using coculture of NFAT-luciferase Jurkat cells with CLDN18.2+ SNU620 cells for 6 hours. To do so, 1×105 Jurkat NFAT-luciferase reporter cells and 1×105 CLDN18.2-expressing NSU620 tumor cells were seeded in a 96-well plate and incubated in 100 μL media+10% FBS and a serial dilution of anti-CEA/CD3 bsAbs at 37° C. for 6 hours. Bio-Glo™ Reagent (Promega) was added and luminescence was quantified using the BioMax Discover system. The data were fitted to a 4PL curve using GraphPad Prism 8 software.
NFAT activity mediated by the 15 humanized CLDN18.2-13/CD3-p subclones, m-13/CD3-p, and Amgen CLDN18.2/CD3 bsAbs (AMG-910) were compared based on the luminescence readout. As expected, humanized CLDN18.2-13/CD3-p subclones induced a similar level of NFAT activity as their parental m-13/CD3-p (
2.4 Anti-Tumor Effect of CLDN18.2-13/CD3 Bispecific Antibodies (i.e. h-13/CD3 bsAbs) with CD3-p (i.e. CD3 Parental) Arm
Finally, we compared the anti-tumor effect of humanized CLDN18.2-13/CD3 subclones (i.e.h-13/CD3-p subclones) with m-13/CD3-p. CLDN18.2 over-expressing HEK/Luc cells (i.e. CLDN18.2+HEK) (ATCC, CRL-3216) and medium CLDN18.2 expressing SNU620 cells were cocultured with PBMCs in serial dilutions of the tested antibodies at a final E/T ratio of 20:1. Specific lysis of tumor cells was determined after a 48-hour coculture by quantification of luciferase intensity units. As shown in
2.5 Binding Affinity of CLDN18.2-13/CD3 Bispecific Antibodies (i.e. h-13/CD3 bsAbs) with Mutant CD3 Arm (CD3-V1 or CD3-V2) to CLDN18.2 and CD3
Since h-13/CD3-p carried SP34 as a CD3 arm, which has high binding affinity to CD3, potential cytokine release syndrome (CRS) may be induced in the clinic. To generate safe and effective CLDN18.2/CD3 bispecific antibody and to reduce the potential CRS related toxicity, we used our optimized CD3 platform, which includes several CD3 binders with various binding affinities. Our previous project has already demonstrated that the usage of the lower binding CD3 binders as arms for construction of CEA/CD3 bispecific antibodies can significantly reduce cytokine release and still retain a potent killing activity.
Therefore, the humanized CLDN18.2-13 heavy chain 1 (VH1) was used to co-transfect CHO cells with h-13 light chains (VL1) linked with the CD3-P, or mutant CD3 binders (CD3-V1 and CD3-V2) (sequences are listed in Table 3). Eventually, three h-13/CD3 variant bispecific antibodies were generated, h-13VH1-VL1/CD3-p, h-13VH1-VL1/CD3-v1, and h-13VH1-VL1/CD3-v2. As the mutant CD3 scFv was linked to hCLDN18.2 light chain, we first examined whether binding affinity of h-13/CD3-v1 and h-13/CD3-v2 to CLDN18.2 on the cell surface would be affected. Therefore, CLDN18.2 positive SNU620 cells were stained with h-13/CD3 variant bispecific antibodies by FACS assay. As shown in
To confirm binding of CD3 variants to CD3 on T cells, we performed a Jurkat binding assay. As we expected, FACS analysis showed that h-13/CD3-v1 and h-13/CD3-v2 displayed significantly reduced binding affinities compared to h-13/CD3-p with parental CD3 binders (i.e.m-13/CD3-p) (
2.6 TCR Signaling Mediated by of CLDN18.2-13/CD3 Bispecific Antibodies (i.e. h-13/CD3 bsAbs) with Mutant CD3 Arm (CD3-V1 or CD3-V2)
We then further evaluated the strength of TCR signaling mediated by h-13/CD3 bsAbs by NFAT reporter assay. As described in section 2.3, Jurkat NFAT-luciferase reporter cells and CLDN18.2-expressing NSU620 tumor cells were seeded in a 96-well plate and incubated for 6 hours. Luminescence was quantified using the BioMax Discover system.
Consistent with their binding affinity (
2.7 Cytolytic Potency Mediated by CLDN18.2-13/CD3 Bispecific Antibodies (i.e. h-13/CD3 bsAbs) with Mutant CD3 Arm (CD3-V1 or CD3-V2)
As CLDN18.2-13/CD3-v2 has lower binding affinity to CD3, which will significantly reduce cytokine release, three CLDN18.2/CD3-v2 bispecific antibodies with VH1 plus different VL1-3 chains were generated. As a result, we further compared cytolytic potency mediated by the 3 h-13/CD3 subclones using various CLDN18.2-expressing HEK (i.e. CLDN18.2+HEK), SNU620 and KATO III cells as a tumor target (
2.8 Cytokine Release Level Mediated by CLDN18.2-13/CD3 Bispecific Antibodies (i.e. h-13/CD3 bsAbs) with Mutant CD3 Arm (CD3-V1 or CD3-V2)
Moreover, since cytokine release level is usually correlated with killing potency, we further compared the cytokine release from the supernatants of SNU620 and PBMCs coculture system.
As described in Section 1.6, the supernatants of SNU620 and PBMC coculture experiments were collected for cytokine release measurement after 48 hours. IFN-γ, TNF-α, IL-10, and IL-2 release were quantified using BD optEIA human IFNγ and TNFα ELISA kits. Briefly, ELISA plates were coated with capture antibodies overnight. The supernatants were diluted at 1:20 with ELISA buffer and detector antibodies were added after 2 hour-incubation. ELISA substrate was added and the plates were read at 450 nM in SpectraMax reader after HRP was added into each well. Interestingly, h-13VH1-VL1/CD3-v2 induced comparable IFN-γ, TNF-α, IL-10, and IL-2 release as the other two h-13/CD3-v2 subclones (
This application claims the benefit of U.S. Provisional Patent Application No. 63/147,233, entitled “CLDN18.2/CD3 bispecific antibodies for the therapy of CLDN18.2-expressing solid tumors”, filed on Feb. 8, 2021, the content of which is herein incorporated by reference in its entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2022/075326 | 2/7/2022 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63147233 | Feb 2021 | US |
