CN114057882B

CN114057882B - Multivalent multispecific antibodies

Info

Publication number: CN114057882B
Application number: CN202110794916.9A
Authority: CN
Inventors: 周立; 阿瓦尼许.瓦许尼; 哈沙尔.帕特尔; 依瑞娜.施耐德
Original assignee: Shandong Boan Biotechnology Co Ltd
Current assignee: Shandong Boan Biotechnology Co Ltd
Priority date: 2020-07-31
Filing date: 2021-07-14
Publication date: 2023-01-06
Anticipated expiration: 2041-07-14
Also published as: CN114057882A

Abstract

The present invention relates to a multivalent multispecific antibody comprising two heavy chains, two light chains and fifth and sixth antigen binding fragments, (1) each of said heavy chains comprises a first and second antigen binding fragment and a heavy chain constant region, each of said first and second antigen binding fragments being directly or indirectly linked to said heavy chain C _H 1 domain N-terminal; and (2) each of the light chains comprises third and fourth antigen binding fragments and a light chain constant region, each of the third and fourth antigen binding fragments being directly or indirectly linked to the N-terminus of the light chain constant region; and (3) the fifth and sixth antigen-binding fragments are each directly or indirectly linked to the C-terminus of the light chain constant region. The multivalent multispecific antibody provided by the invention has obviously improved affinity of tumor cell surface antigen and obviously improved tumor cell killing power.

Description

Multivalent multispecific antibodies

Technical Field

The present application relates to multivalent multispecific antibodies, in particular to tetravalent bispecific antibodies comprising specific binding to receptor tyrosine kinase-like orphan receptor 1 (ROR 1) and CD3. The application also relates to polynucleotides encoding said antibodies, host cells comprising said polynucleotides, methods of producing said antibodies and therapeutic uses thereof.

Background

CD3 bispecific T cell adaptors are one of the most promising bispecific antibody platforms in the development of effective cancer therapy areas. One part of the antibody binds to the tumor antigen and the other part binds to CD3 of the TCR, engaging the T cell in killing the cancer cell. Tumor-redirecting T-cell killing is a new function that is not present in the parent antibody used to make the T-cell adaptor antibody, which cannot be replaced by a mixture of the two parent antibodies, and is therefore called a mandatory Bispecific antibody (Bispecial antibodies: labrijn AF, a mechanistic review of the pipeline. Janmaat ML, reichert JM, parren PWHI. Nat Rev Drug discovery. 2019 Aug;18 (8): 585-608) (T cell encoding Bispecific antibody (T-BsAb): from technology to therapy. Wu Z, chemistry NV. Pharmacol. Ther.2018 Feb; 182.

One of the major bottlenecks in the development of CD 3T cell adaptors is the possible toxicity at higher therapeutically effective dose levels due to the high release of cytokines. Another bottleneck is that most tumor antigens are also expressed on normal tissues/cells, which, although at a lower level, also leads to a certain targeted toxicity. The development of multivalent (e.g., tetravalent) tumor antigen binding molecules will more specifically enhance binding to tumor cells with higher antigen expression than to normal cells with lower antigen expression, thereby increasing therapeutic index. Multivalent (preferably tetravalent) tumor cell binding may be (1) to the same epitope of the same antigen of interest, (2) to the same antigen of interest to different, non-overlapping epitopes (double complementary epitopes), or (3) to different antigens of interest (double targeting).

Receptor tyrosine kinase-like orphan receptor 1 (ROR 1) is a member of the ROR family consisting of ROR1 and ROR 2. It comprises two different cysteine-rich extracellular domains and a transmembrane domain. In the intracellular portion, ROR1 possesses a tyrosine kinase domain, two serine/threonine rich domains and a proline rich domain. Much like the physiological function of ROR1, ROR1 may also have a kinase activity dependent or independent function in cancer, which may be the result of tissue specific expression of co-receptors or effector proteins. The notion that ROR1 knockdown induces apoptosis, EGFR signaling potentiation, and ROR 1-mediated upregulation of epithelial-mesenchymal transition (EMT) genes supports the important role ROR1 plays in cancer progression. In another mechanism, expression of ROR1 on tumor cells activates Wnt5a signaling, a pathway important for tumor cell proliferation, migration and survival. Upregulation of ROR1 is commonly detected in hematological and solid tumors, which has led to a high scientific interest in exploring the functional advantage of ROR1 expression in targeted cancer cells as a therapeutic strategy. Various studies have developed monoclonal antibodies against ROR1 and shown that therapeutic efficacy is obtained directly by ADCC or CDC without any effect on apoptosis of ROR1 negative cell lines. There are also a number of studies involving the transduction of patient T cells with ROR1 Chimeric Antigen Receptors (CARs). ROR1-CAR T cells can recognize tumor cells and lyse primary tumor cells. Different stages of clinical studies are currently being conducted to evaluate the efficacy of a treatment targeting ROR1.

The Claudin family of proteins has at least 27 member molecules in mammals (Furute M.et al.J Cell biol.,1998,141,1539). These tight junction molecules are indispensable for the paracellular barrier in the vertebrate epithelial cell sheet. The Claudin 18 molecule is an intact transmembrane protein containing four transmembrane hydrophobic regions and two extracellular loops. There are two different splice variants of Claudin 18. Subtype 1 (Claudin 18.1 or CLDN 18.1) is selectively expressed on cells of normal lung tissue, and subtype 2 (Claudin 18.2 or CLDN 18.2) is considered to be a cancer-associated splice variant (Sanada y.et al.j pathol.,2006,208,633).

Claudin 18.2 is a CD 20-like differentiation protein that is overexpressed in non-small cell lung cancers (NSCLCs; 25%), gastric cancers (70%), pancreatic cancers (50%) and esophageal cancers (30%). The expression of this protein is influenced by the ethnic background of the patient. For example, the expression level is higher in Japanese patients compared to caucasian patients. Claudin 18.2 is also ectopically expressed in ovarian, breast and head and neck tumors (Sahin u.et al. Clin Cancer res.2008,14,7624). Claudin 18.2 can be detected in lymph nodes and distant metastasis from gastric carcinoma. Its expression in normal tissues is strictly restricted to the short-lived epithelial cells of the gastric mucosa.

Claudin 18.2 is likely to play a complex role in tumorigenesis and maintenance of the tumor microenvironment. It was found that EGFR/ERK signaling induces biliary tumor-associated Claudin 18 expression, and is involved in cell proliferation, invasion and tumorigenicity in vivo (Kumi, t., cancer lett.,2017,403,66). Claudin-18 has also been reported to inhibit abnormal proliferation and motility of lung epithelial cells by inhibiting the PI3K/PDK1/AKT signaling pathway (Shun, S.et al.Bio Bioph Acta (BBA) -Mobile Cell Res,2016,1863,1170). In mice with the Claudin 18.2 knock-out, the paracellular barrier is compromised, resulting in leakage of H + ions secreted by gastric parietal cells through the gastric epithelial layer into the gastric lumen, followed by a decrease in the pH of the stomach. Chronic gastritis in knockout mice results in high levels of inflammatory cells and metaplastic (SPEM) cells expressing spastic polypeptides. Inflammation caused by inflammatory cells is characterized by higher expression of various proinflammatory markers, such as IL-1 β and TNF- α (Hayashi, d.gastroenterology,2012,142,292).

Although the biological function of Claudin 18.2 in tumorigenesis and tumor microenvironment is uncertain, it is clear that Claudin 18.2 is expressed in gastric Cancer transformation and is abnormally activated in a variety of tumors, including esophageal, pancreatic and biliary tract cancers (Micke, p.et al., intl J Cancer,2014,135,2206). The exposed extracellular loop of Claudin 18.2 allows for binding of monoclonal antibodies, and appropriate antibodies that bind to Claudin 18.2 on the surface of tumor cells can kill tumor cells by antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) effects. anti-Claudin 18.2 compounds can also induce apoptosis and inhibit cell proliferation. When combined with chemotherapy, they may enhance T cell infiltration and induce pro-inflammatory cytokines.

Thus, claudin 18.2 is a valuable target for the prevention and/or treatment of several primary tumors, e.g. gastric, esophageal, pancreatic, lung cancer such as non-small cell lung cancer, ovarian, colon, liver, head and neck and gall bladder cancer and metastases thereof, in particular gastric cancer metastases such as kunkenberg, peritoneal and lymph node metastases.

It is an object of the present application to provide a novel multivalent multispecific antibody with particularly advantageous properties, such as improved binding affinity and improved cytotoxic potency.

Disclosure of Invention

The present invention provides novel multivalent multispecific antibodies with particularly advantageous properties, such as improved binding affinity and improved cytotoxic potency.

In one aspect, the invention provides a multivalent multispecific antibody comprising two heavy chains, two light chains, and fifth and sixth antigen-binding fragments,

(1) Wherein each of the heavy chains comprises first and second antigen-binding fragments and a heavy chain constant region, wherein the first and second antigen-binding fragments are directly or indirectly linked to C _H 1 domain N-terminal; and

(2) Wherein each of the light chains comprises third and fourth antigen-binding fragments and a light chain constant region (such as C) _κ Or C _λ ) Wherein the third and fourth antigen binding fragments are directly or indirectly linked to the N-terminus of the light chain constant region; and

(3) Wherein each of the fifth and sixth antigen-binding fragments is directly or indirectly linked to the C-terminus of the light chain constant region.

In another aspect of the invention, the first and second antigen-binding fragments on the heavy chain and the third and fourth antigen-binding fragments on the light chain bind to the same epitope of the antigen.

In another aspect of the invention, the first and second antigen binding fragments on the heavy chain bind to one epitope of an antigen and the third and fourth antigen binding fragments on the light chain bind to another epitope of the antigen.

In another aspect of the invention, the first antigen-binding fragment on the heavy chain binds to a first epitope of an antigen, the second antigen-binding fragment on the heavy chain binds to a second epitope of the antigen, the third antigen-binding fragment on the light chain binds to a third epitope of the antigen, and the fourth antigen-binding fragment on the light chain binds to a fourth epitope of the antigen.

In another aspect of the invention, the first antigen-binding fragment binds to a first antigen, the second antigen-binding fragment binds to a second antigen, the third antigen-binding fragment binds to a third antigen, and the fourth antigen-binding fragment binds to a fourth antigen.

In another aspect of the invention, the first and second antigen-binding fragments on the heavy chain bind to a first antigen and the third and fourth antigen-binding fragments on the light chain bind to a second antigen.

In another aspect of the invention, the antigen is selected from tumor target antigens of hematological tumors and solid tumors.

In another aspect of the invention, the antigen is one or more of CEA, claudin 18.2, GPC3, receptor tyrosine kinase-like orphan receptor 1 (ROR 1), CD38, her2, CD19, CD20, CD22, BCMA, CAIX, CD446, CD133, EGFR, EGFRvIII, epCam, GD2, ephA2, her1, her2, ICAM-1, IL13Ra2, mesothelin, MUC1, MUC16, NKG2D, PSCA, NY-ESO-1, MART-1, WT1, MAGE-A10, MAGE-A3, MAGE-A4, EBV, NKG2D, PD, PD-L1, CD25, IL-2, and CD3.

In another aspect of the invention, the first and second antigen-binding fragments on the heavy chain and the third and fourth antigen-binding fragments on the light chain bind to ROR1.

In another aspect of the invention, the first and second antigen-binding fragments on the heavy chain bind to one epitope of ROR1, and the third and fourth antigen-binding fragments on the light chain bind to another epitope of ROR1.

In another aspect of the invention, the first and second antigen-binding fragments on the heavy chain bind to ROR1 and the third and fourth antigen-binding fragments on the light chain bind to Claudin 18.2.

In another aspect of the invention, the first and second antigen-binding fragments on the heavy chain bind to Claudin 18.2 and the third and fourth antigen-binding fragments on the light chain bind to ROR1.

In another aspect of the invention, the ROR1 is human ROR1.

In another aspect of the invention, said Claudin 18.2 is human Claudin 18.2.

In another aspect of the invention, the fifth and sixth antigen binding fragments on the light chain bind to CD3.

In another aspect of the invention, the CD3 is human CD3.

In another aspect of the invention, each of the heavy chain constant regions comprises C _H 1,C _H 2 and C _H 3, or C _H 1,C _H 2,C _H 3 and C _H 4。

In another aspect of the invention, the light chain constant region is C _κ Or C _λ And (4) molding.

In another aspect of the invention, the first, second, third, fourth, fifth and sixth antigen-binding fragments are selected from the group consisting of scFv fragments, fv fragments, F (ab') ₂ Fragments, fab' fragments, VHH, V _NAR Or SD (single domain).

In another aspect of the invention, the first, second, third and fourth antigen binding fragments are VHHs.

In another aspect of the invention, the fifth and sixth antigen-binding fragments are scfvs.

In another aspect of the invention, the fifth and sixth antigen-binding fragments are scfvs and are linked to the C-terminus of the light chain constant region by a linker.

In another aspect of the invention, the linker is (G) ₄ S) ₃ A linker.

In another aspect of the invention, the first, second, third and fourth antigen-binding fragments are the same VHH that specifically binds to human ROR1, said VHH having CDR1-CDR3 shown in SEQ ID Nos. 18-20; more preferably, the VHH comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence of SEQ ID No. 3.

In another aspect of the invention, the third and fourth antigen binding fragments are the same SD (single domain) that specifically binds to human Claudin 18.2, wherein SD comprises CDR1-CDR3 as set forth in SEQ ID NOS: 21-23; preferably, the SD comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence of SEQ ID No. 4.

In another aspect of the invention, the first and second antigen-binding fragments are the same VHH that specifically binds to human ROR1, and wherein the VHH has CDR1-CDR3 shown in SEQ ID NOs 18-20; more preferably, the VHH comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence of SEQ ID No. 3, and wherein the third and fourth antigen-binding fragments are the same SD (single domain) that specifically binds to human Claudin 18.2, and wherein SD comprises CDR1-CDR3 as set forth in SEQ ID NOs 21-23; preferably, the SD comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence of SEQ ID No. 4.

In another aspect of the invention, the first and second antigen-binding fragments are the same SD (single domain) that specifically binds to human Claudin 18.2, and wherein the SD comprises CDR1-CDR3 as set forth in SEQ ID NOS: 21-23; preferably, the SD comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence of SEQ ID No. 4.

In another aspect of the invention, the first and second antigen-binding fragments are the same SD (single domain) that specifically binds to human Claudin 18.2, and wherein the SD comprises CDR1-CDR3 as set forth in SEQ ID NOS: 21-23; preferably, the SD comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence of SEQ ID No.4, and wherein the third and fourth antigen-binding fragments are the same VHH that specifically binds to human ROR1, and wherein the VHH has CDR1-CDR3 as set forth in SEQ ID NOs 18-20; more preferably, the VHH comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence of SEQ ID No. 3.

In another aspect of the invention, the first, second, third and fourth antigen binding fragments are the same SD (single domain) that specifically binds to human Claudin 18.2, and wherein SD comprises CDR1-CDR3 as set forth in SEQ ID NOS: 21-23; preferably, the SD comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence of SEQ ID No. 4.

In another aspect of the invention, the fifth and sixth antigen-binding fragments are scFvs and each comprise a light chain variable region comprising CDR1-CDR3 as set forth in SEQ ID NOs 15-17; preferably, the light chain variable region comprises an amino acid sequence having at least about 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO. 2 and/or the heavy chain variable region comprises CDR1-CDR3 of SEQ ID NO. 12-14; preferably, the light chain variable region comprises an amino acid sequence having at least about 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO: 1.

In another aspect of the invention, the fifth and sixth antigen-binding fragments are scFvs and each comprise a light chain variable region comprising an amino acid sequence having at least about 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO. 7 and/or a heavy chain variable region comprising a modified version of SEQ ID NO.1,

preferably, the modified version of SEQ ID NO.1 has one or more mutations selected from N30S, K31T, F W, K bN and Y58T; preferably, the modified version has any one of the following 1) to 3): 1) The N30S, K T and F98W mutations; 2) N30S, K T, F W and K52bN mutations; 3) N30S, K T, F W and Y58T mutations, numbering system Kabat numbering system; or alternatively

1 has one or more mutations selected from the group consisting of Q13K, K R and L108T; preferably, the modified version has the Q13K, K R and L108T mutations, numbering system is Kabat numbering system;

more preferably, the heavy chain variable regions of said fifth and sixth antigen binding fragments comprise amino acid sequences having at least about 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequences of SEQ ID No.8 or SEQ ID No.9 or SEQ ID No.10 or SEQ ID No. 11.

In another aspect of the invention, there is provided a nanobody (VHH) comprising CDR1-CDR3 shown in SEQ ID NOS: 18-20; preferably, the nanobody comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence of SEQ ID No. 3.

In another aspect of the invention, there is provided a Single Domain (SD) binding fragment comprising the CDR1-CDR3 shown in SEQ ID NOS: 21-23; preferably, the single domain binding fragment comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence of SEQ ID NO. 4.

In another aspect of the invention, there is provided a polynucleotide encoding said multivalent multispecific antibody or said nanobody or said Single Domain (SD) binding fragment.

In another aspect of the invention, there is provided a vector comprising said polynucleotide.

In another aspect of the invention, there is provided a host cell comprising said multivalent multispecific antibody, said nanobody, said Single Domain (SD) binding fragment, said polynucleotide, or said vector.

In another aspect of the invention, the host cell is a mammalian cell, a fungal cell, a plant cell or a bacterium.

In another aspect of the invention, the host cell is a human cell.

In another aspect of the invention, there is provided a pharmaceutical composition comprising said multivalent multispecific antibody, said nanobody, said Single Domain (SD) binding fragment, said polynucleotide, said vector, or said host cell.

In another aspect of the invention, the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients.

In another aspect of the invention, there is provided a use of the multivalent multispecific antibody, the nanobody, the Single Domain (SD) binding fragment, the polynucleotide, the vector, or the host cell in the manufacture of a medicament for the treatment of cancer in a subject.

In another aspect of the invention, there is provided a method of treating cancer in a subject in need thereof, comprising administering to the subject the multivalent multispecific antibody, the nanobody, the Single Domain (SD) binding fragment, the polynucleotide, the vector, or the host cell.

In another aspect of the invention, the cancer is a hematological tumor or a solid tumor.

In another aspect of the invention, the cancer is a ROR 1-positive or Claudin 18.2-positive cancer.

In another aspect of the invention, wherein the cancer is selected from one or more of ovarian cancer, pancreatic cancer, colon cancer, colorectal cancer, lymphoma, esophageal cancer, lung cancer, ovarian cancer, liver cancer, head and neck cancer, and gallbladder cancer.

The scheme of the invention has the following advantages:

1) The binding affinity of the tetravalent anti-ROR 1-CD3 bispecific antibody (115-tetravalent-CD 3) provided by the invention and the human ROR1 protein or the tumor cell line expressing the ROR1 protein is obviously higher than that of the bivalent anti-ROR 1 antibody (115-bivalent).

2) The tetravalent anti-ROR 1-CD3 bispecific antibody (115-tetravalent-CD 3) provided by the invention can effectively kill tumor cells by engaging T cells, and has remarkably improved tumor cell killing power compared with 115-divalent antibody and 115/HC-841/LC-SP34 antibody.

3) The tetravalent anti-ROR 1-CD3 bispecific antibody (115-tetravalent-CD 3) has a higher affinity for tumor targets than the divalent-CD 3 antibody, i.e. the divalent 841-SP34 bispecific antibody, and results in more efficient lysis of tumor cells.

4) When the mutant anti-CD 3 antigen binding fragment is adopted in the tetravalent anti-ROR 1-CD3 bispecific antibody (115-tetravalent-CD 3), the affinity of the antibody to CD3 can be reduced, and the release of cytokines can be reduced.

Drawings

The novel features believed characteristic of the invention are set forth with particularity in the appended claims. In order that the features and advantages of the present application may be better understood, the present application will be described in detail in the following examples and examples.

FIG. 1A shows a schematic representation of a tetravalent anti-ROR 1-CD3 bispecific antibody (115-tetravalent-CD 3) of the present application; FIG. 1B shows the detailed structure from N-terminus to C-terminus of the heavy and light chains of the tetravalent anti-ROR 1-CD3 bispecific antibody.

FIG. 2A shows a schematic of a construct of a bivalent anti-ROR 1 antibody (115-bivalent); FIGS. 2B-2D show binding of tetravalent anti-ROR 1-CD3 bispecific antibody (115-tetravalent-CD 3), bivalent anti-ROR 1 antibody (115-bivalent) to human ROR1 and human CD3e ELISAs.

FIGS. 3A-3C show binding of tetravalent anti-ROR 1-CD3 bispecific antibody (115-tetravalent-CD 3), divalent anti-ROR 1 antibody (115-divalent) to ROR1 on cells in different tumor cell lines.

FIG. 4A shows a schematic of an isotype control anti-IC/SP 34 bispecific antibody; FIGS. 4B-4D show the killing assay of a tetravalent anti-ROR 1-CD3 bispecific antibody (115-tetravalent-CD 3) against ROR 1-expressing tumor cells.

FIG. 5A shows a schematic diagram of the 115/HC-841/LC-SP34 antibody; FIG. 5B shows a schematic representation of a bivalent 841-SP34 bispecific antibody; FIG. 5C shows a comparison of binding of tetravalent anti-ROR 1-CD3 bispecific antibody (115-tetravalent-CD 3), divalent 841-SP34 bispecific antibody, 115/HC-841/LC-SP34 antibody to human ROR 1; FIG. 5D shows the binding comparison of tetravalent anti-ROR 1-CD3 bispecific antibody (115-tetravalent-CD 3), divalent 841-SP34 bispecific antibody, 115/HC-841/LC-SP34 antibody with human CD3 e.

FIG. 6A shows the killing activity of tetravalent anti-ROR 1-CD3 bispecific antibody (115-tetravalent-CD 3), 115/HC-841/LC-SP34 antibody, 115-bivalent antibody against ROR1 positive MDA-MB-231 cells; FIG. 6B shows the killing activity of tetravalent anti-ROR 1-CD3 bispecific antibody (115-tetravalent-CD 3), 115/HC-841/LC-SP34 antibody, 115-bivalent antibody against ROR 1-positive HT-29 cells; FIG. 6C shows the killing activity of tetravalent anti-ROR 1-CD3 bispecific antibody (115-tetravalent-CD 3), 115/HC-841/LC-SP34 antibody, 115-divalent antibody against MDA-MB-231 cells and HT-29 cells.

Detailed Description

It is understood that different applications of the disclosed products and methods may be tailored to specific needs in the art. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only and is not intended to be limiting.

Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly used in the art to which this invention belongs. For the purpose of interpreting the specification, the following definitions will apply and where 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" are intended to include the plural forms as well, unless the context clearly indicates otherwise, e.g., reference to "a host cell" includes a plurality of such host cells.

Detailed description of the sequences of the present application

Table 1 (CDR region in italics, IMGT system for identification system)

Example 1

Generation of tetravalent anti-ROR 1-CD3 bispecific antibody (115-tetravalent-CD 3, or 115-tetravalent-SP 34), divalent anti-ROR 1 antibody (115-divalent)

The constructs of tetravalent anti-ROR 1-CD3 bispecific antibodies in the present application are designed to link an antigen binding fragment (VHH) that specifically binds the ROR1 antigen expressed on tumor cells on the N-terminus of the CH1 and CL domains of the antibody and a single chain antibody (scFv) against CD3 on the C-terminus of the CL domain of the antibody, as shown in fig. 1A; the detailed structure of the heavy chain and the light chain of the tetravalent anti-ROR 1-CD3 bispecific antibody from N end to C end is shown in FIG. 1B.

The construct of the bivalent anti-ROR 1 antibody (115-bivalent) with the configuration shown in fig. 2A (115-bivalent) was designed to attach an antigen binding fragment (VHH) that specifically binds to ROR1 antigen expressed on tumor cells to the N-terminus of the CH1 domain of the antibody, and did not contain a light chain.

The VHH antibody aiming at ROR1, namely a nano antibody, is obtained by screening and separating a full-length human ROR1 protein from an alpaca by using a phage display technology platform after the alpaca is immunized by using a full-length human ROR1 (NP-001077061.1) antigen. The sequences of the selected VHH are shown in table 2. The heavy chain variable region (VH) and light chain variable region (VL) of the anti-CD 3 single chain antibody (scFv) were derived from the humanized IgG antibody SP34, and the VH and VL sequences are shown in table 2. VH and VL sequences were generated by gene synthesis from GeneArt AG (Thermo Fisher Scientific, regensburg, germany).

The sequences used for the tetravalent anti-ROR 1-CD3 bispecific antibody (115-tetravalent-CD 3) and the divalent anti-ROR 1 antibody (115-divalent) in this example are shown in Table 2; the tetravalent anti-ROR 1-CD3 bispecific antibody (115-tetravalent-CD 3) and the bivalent anti-ROR 1 antibody (115-bivalent) were generated by gene synthesis from GeneArt AG (Thermo Fisher Scientific, regensburg, germany) according to the sequences in Table 2.

TABLE 2

Example 2

The tetravalent anti-ROR 1-CD3 bispecific antibody (115-tetravalent-CD 3) has higher binding affinity for human ROR1 and CD3 epsilon (i.e., human CD3 e) proteins than the divalent anti-ROR 1 antibody (115-divalent).

To evaluate the binding activity of constructs 115-bivalent and 115-tetravalent-CD 3 antibodies to human ROR1 and CD3 epsilon proteins, a protein-based ELISA assay was performed. 96 well ELISA plates were coated with 100. Mu.L of 1. Mu.g/mL human-ROR 1-His (Acro Biosystems) or 0.5. Mu.g/mL human-CD 3 epsilon-His protein (Acro Biosystems) overnight at 4 ℃. Plates were blocked with 2% BSA/PBS for 1 hour at room temperature, followed by serial dilutions of test antibody: a tetravalent anti-ROR 1-CD3 bispecific antibody (115-tetravalent-CD 3) or a divalent anti-ROR 1 antibody (115-divalent) was incubated at room temperature for 1 hour. After washing the plate with PBS-T, 100. Mu.L of Peroxidase Affini Pure Goat Anti-Human IgG (Peroxidase Affinipure Goat Anti-Human I)gG), fc gamma fragment specificity (PBST 1: 5000 dilution) at room temperature incubation plate for 30 minutes. ELISA substrate TMB (BD Biosciences) was added using 2N H ₂ SO ₄ The reaction was stopped. The absorbance was read at 450nm with a SpectraMax plate reader.

As shown in FIGS. 2B-2D, the binding affinity of the tetravalent anti-ROR 1-CD3 bispecific antibody (115-tetravalent-CD 3) to human ROR1 was about 12 times that of the divalent anti-ROR 1 antibody (115-divalent). For human CD3 epsilon (i.e., human CD3 e), only binding of the tetravalent anti-ROR 1-CD3 bispecific antibody (115-tetravalent-CD 3) was observed.

Example 3

Tetravalent anti-ROR 1-CD3 bispecific antibody (115-tetravalent-CD 3), bivalent anti-ROR 1 antibody (115-bivalent) bind to ROR1 on ROR1 expressing tumor cell lines

Fluorescence Activated Cell Sorting (FACS) assay further confirmed the binding of tetravalent anti-ROR 1-CD3 bispecific antibody (115-tetravalent-CD 3), divalent anti-ROR 1 antibody (115-divalent) to ROR1 on ROR1 expressing tumor cell lines.

Target tumor cells (HPAC, HT-29 or JeKo 1) with different ROR1 expression levels at 1X10 ⁵ In a 96-well non-adherent tissue culture plate. Serially diluted antibodies (115-tetravalent-CD 3 or 115-divalent) were added to the wells and incubated at 4 ℃ for 1 hour. After washing the plates with FACS wash buffer, the plates were incubated with PE-conjugated goat anti-human IgG Fc fragment specific antibody (1. Mean Fluorescence Intensity (MFI) was measured by flow cytometry and the results were analyzed using GraphPad Prism software.

The results of this experiment show that the tetravalent anti-ROR 1-CD3 bispecific antibody (115-tetravalent-CD 3) binds more strongly to ROR1 on these tumor cell lines than to ROR1 on these tumor cell lines at a moderate level of binding of the bivalent anti-ROR 1 antibody (115-bivalent) to ROR1, as shown in FIGS. 3A-3C.

Example 4

Tetravalent anti-ROR 1-CD3 bispecific antibody (115-tetravalent-CD 3) has cytotoxic effect on T cell involvement and mediation of ROR 1-expressing tumor cells

Killing in activated human Peripheral Blood Mononuclear Cells (PBMCs)In the experiments, the involvement of T cells and the killing of antigen-specific tumor cells was investigated. Human PBMCs were freshly isolated from leukocytes (Leukopak) from healthy donors and activated after 6 days of culture with anti-CD 3/CD28 coated beads. Activated T cells (E/T ratio 10: 1) were then co-incubated with tumor target cell lines (HPAC or HT29, 10000 cells per well) with different ROR1 expression levels in the presence of a tetravalent anti-ROR 1-CD3 bispecific antibody (115-tetravalent-CD 3) or an isotype control anti-IC/SP 34 bispecific antibody (binding to IC and CD3, fig. 4A shows the configuration of the isotype control anti-IC/SP 34 bispecific antibody, the sequences of the anti-IC VH and VL are shown in table 3, binding to antigen IC on the surface of non-tumor cells). CO at 37 ℃ and 5% ₂ After 48 hours of incubation, the lethality of the tetravalent anti-ROR 1-CD3 bispecific antibody (115-tetravalent-CD 3) or isotype control anti-IC/SP 34 bispecific antibody to the target cells was evaluated by evaluating luciferase signal based on the average Relative Light Units (RLU) per sample well. Maximal luciferase signal of the target cells was achieved by incubating the target cells without bispecific antibody (i.e. only target cells and activated T cells) (this example allows the target cells themselves to produce luciferase signal, in the absence of antibody, the target cells are not killed, luciferase signal is maximal, survival rate is 100%). Survival (survival of viability) was calculated as RLU (antibody)/RLU (no antibody control) x 100. As shown in FIGS. 4B-4D, the tetravalent anti-ROR 1-CD3 bispecific antibody (115-tetravalent-CD 3) has higher cytotoxic potency with EC50 of 1.04nM and 0.29nM, respectively, against ROR 1-expressing HPAC and HT29 tumor cell lines.

TABLE 3

Example 5

The tetravalent anti-ROR 1-CD3 bispecific antibody (115-tetravalent-CD 3, or 115-tetravalent-SP 34) has a higher binding affinity than the divalent 841-SP34 bispecific antibody

It is expected that the tetravalent anti-ROR 1-CD3 bispecific antibody (115-tetravalent-CD 3) should potentially have a higher affinity for tumor targets and result in more efficient lysis of tumor cells than the divalent-CD 3 antibody, i.e., the divalent 841-SP34 bispecific antibody.

To verify this expectation, a 115/HC-841/LC-SP34 antibody was obtained using a single domain antibody specific for Claudin 18.2 (single domain antibody SD of sequence SEQ ID No.4, table 4, which was designed from a VH-VL complex that binds Claudin 18.2 as a single domain antibody against Claudin 18.2 antibody (clone 841)) instead of 115-VHH on the tetravalent anti-ROR 1-CD3 bispecific antibody light chain, the configuration being shown in FIG. 5A, this construct also being obtained in a manner similar to that described in example 1.

A bivalent 841-SP34 bispecific antibody was constructed, which was an anti-Claudin 18.2/CD3 bispecific antibody, designed by linking the VH and VL sequences of anti-Claudin 18.2 at the N-terminus of the CH1 and CL domains of the antibody (VH and VL sequences are shown in SEQ ID Nos. 5-6, table 4), and by linking an anti-CD 3 single chain antibody (scFv) (VH and VL sequences in anti-CD 3 single chain antibody (scFv) are shown in SEQ ID Nos. 1-2) at the C-terminus of the CL domain of the antibody, in the configuration shown in FIG. 5B.

To assess binding activity to human ROR1 or human CD3e protein, protein-based ELISA assays are provided as in example 2. As expected, the tetravalent anti-ROR 1-CD3 bispecific antibody (115-tetravalent-CD 3) was 4 times higher than the binding affinity of the 115/HC-841/LC-SP34 antibody for human ROR1, and the divalent 841-SP34 bispecific antibody did not show any binding to human ROR1 (FIG. 5C). Unexpectedly, the 115/HC-841/LC-SP34 antibody reduced human CD3e binding affinity to some extent compared to the tetravalent anti-ROR 1-CD3 bispecific antibody (115-tetravalent-CD 3) (FIG. 5D).

TABLE 4

Example 6

Quadrivalence is associated with higher killing of tumor cells

To further confirm whether the 115-tetravalent nature of the antibody is directly related to lethality, we performed standard cytotoxicity assays in two ROR1 positive cell lines (MDA-MB-231 and HT-29 cell lines) as described in example 4. Antibody-mediated cytotoxicity was assessed by luciferase from live MDA-MB-231 or HT-29Luc cells after 48-hour incubation in serial dilutions of antibodies (tetravalent anti-ROR 1-CD3 bispecific antibody (115-tetravalent-CD 3), 115/HC-841/LC-SP34 antibody, 115-bivalent antibody). As shown in fig. 6A, 6B and 6C, the tetravalent anti-ROR 1-CD3 bispecific antibody (115-tetravalent-CD 3) antibody enhanced T cell redirected cytotoxicity of ROR1 expressing tumor cells in a dose-dependent manner with EC50 of 0.027nM (using MDA-MB-231 cell line) and 0.32 (using low ROR1 expressing cell line HT-29). However, a decrease in potency was observed in the treatment with the 115/HC-841/LC-SP34 antibody, with an EC50 of 3.74nM (using the MDA-MB-231 cell line), with 100-fold less lethality compared to the 115-tetravalent-CD 3 antibody. We did not observe a significant lethality of HT-29 cells with 115-bivalent antibody. This data indicates that the 115-tetravalent-CD 3 antibody molecule has a higher cytotoxic potency than the 115-bivalent antibody and the 115/HC-841/LC-SP34 antibody.

It is to be understood that while the present 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.

The various embodiments described above can be combined to provide further embodiments. All 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, if necessary, to employ concepts of the various patents, applications and publications to provide yet further embodiments.

These and other modifications can be made to embodiments of the present invention 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.

Sequence listing

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Claims

1. A multivalent multispecific antibody comprising two heavy chains, two light chains, and fifth and sixth antigen-binding fragments,

(1) Each of the heavy chains comprises first and second antigen-binding fragments each directly or indirectly linked to the heavy chain C and a heavy chain constant region _H 1 domain N-terminal; and

(2) Each of the light chains comprises third and fourth antigen binding fragments and a light chain constant region, each of the third and fourth antigen binding fragments being directly or indirectly linked to the N-terminus of the light chain constant region; and

(3) (iii) the fifth and sixth antigen-binding fragments are each directly or indirectly linked to the C-terminus of the light chain constant region;

the first, second, third and fourth antigen-binding fragments are VHHs that bind to receptor tyrosine kinase-like orphan receptor 1 (ROR 1), the fifth and sixth antigen-binding fragments are scfv that bind to CD 3; the VHH includes CDR1-CDR3 shown in SEQ ID NO. 18-20.

2. The multivalent multispecific antibody of claim 1, wherein the VHH comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence of SEQ ID No. 3.

3. The multivalent multispecific antibody of claim 1, wherein the scfv that binds CD3 comprises a light chain variable region comprising CDR1-CDR3 of SEQ ID NOs 15-17 and a heavy chain variable region comprising CDR1-CDR3 of SEQ ID NOs 12-14.

4. The multivalent multispecific antibody of claim 3, wherein the light chain variable region comprises an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID No. 2 and the heavy chain variable region comprises an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID No. 1.

5. A VHH that specifically binds to human ROR1, comprising CDR1-CDR3 shown in SEQ ID NOs: 18-20.

6. The VHH according to claim 5, which specifically binds to human ROR1, said VHH comprising an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence of SEQ ID NO 3.

7. A polynucleotide encoding a multivalent multispecific antibody according to any one of claims 1 to 4 or a VHH according to any one of claims 5 to 6 that specifically binds to human ROR1.

8. A vector comprising the polynucleotide of claim 7.

9. A pharmaceutical composition comprising a multivalent multispecific antibody according to any one of claims 1 to 4, a VHH according to any one of claims 5 to 6 which specifically binds to human ROR1, a polynucleotide according to claim 7, or a vector according to claim 8.

10. The pharmaceutical composition of claim 9, further comprising one or more pharmaceutically acceptable excipients.

11. Use of a multivalent multispecific antibody according to any one of claims 1 to 4, a VHH which specifically binds to human ROR1 according to any one of claims 5 to 6, a polynucleotide according to claim 7, a vector according to claim 8 or a pharmaceutical composition according to any one of claims 9 to 10 for the manufacture of a medicament for the treatment of a cancer in a subject, which cancer is a ROR 1-positive cancer.

12. The use according to claim 11, wherein the cancer is selected from one or more of ovarian cancer, pancreatic cancer, colon cancer, colorectal cancer, lymphoma, esophageal cancer, lung cancer, ovarian cancer, liver cancer, head and neck cancer, and gallbladder cancer.