WO2018166527A1

WO2018166527A1 - Multispecific antibody, antibody conjugate and related pharmaceutical composition and use

Info

Publication number: WO2018166527A1
Application number: PCT/CN2018/079284
Authority: WO
Inventors: 王�忠; 于浩洋
Original assignee: 本康生物制药（深圳）有限公司
Priority date: 2017-03-17
Filing date: 2018-03-16
Publication date: 2018-09-20
Also published as: CN110520442A

Abstract

Provided are a multispecific antibody targeting the HER2 extracellular domain II and an antigen on an immune effector cell, and a conjugate of the antibody and a nanoparticle, wherein the conjugate can further comprise a binding portion specifically binding to the HER2 extracellular domain IV. Also provided are a pharmaceutical composition comprising the multispecific antibody or the conjugate thereof, and the use of the multispecific antibody or the conjugate thereof in the preparation of a drug for treating HER2-positive tumours.

Description

Multispecific antibodies, antibody conjugates and related pharmaceutical compositions and applications

Field of invention

The present application relates to the field of immunomedicine, and more particularly to bispecific antibodies, and in particular to multispecific antibodies, antibody conjugates and related pharmaceutical compositions and uses that target HER2 extracellular domain II and immune effector cells.

Background of the invention

The human epidermal growth factor receptor (HER) family includes four structurally related members - HER1 (ErbB1, also known as EGFR), HER2 (ErbB2, also known as HER2/neu), HER3 (ErbB3), and HER4 (ErbB4) . HER2 is a 185 kDa protein with an extracellular ligand binding domain (ECD, extracellular domain) and an intracellular tyrosine kinase domain. The N-terminal ECD can be divided into four subdomains (I-IV). HER2 is a promising target for breast cancer treatment because it was found to be overexpressed in approximately one in four breast cancer patients (Bange et al, 2001, Nature Medicine 7: 548). In vitro studies have shown that inhibition of HER2 induces significant apoptosis in breast cancer cells (Faltus T et al, Neoplasia. 2004; 6(6): 786-95; Yang G et al, J Biol Chem. 2004; 279(6): 4339– 45). These studies have led to the development of targeted therapies for HER2-positive breast cancer patients. For example, the first approved HER2 targeted humanized monoclonal antibody trastuzumab (Trastuzumab) (

That is, Herceptin has been identified as an important treatment option for HER2-positive breast cancer. Trastuzumab binds to subdomain IV of HER2 and exerts its anti-tumor via several mechanisms including blocking constitutive HER2 signaling, antibody-dependent cell-mediated cytotoxicity (ADCC), and inhibition of tumor angiogenesis active. However, as a single agent, only 15%-30% of HER2-positive breast cancers respond to trastuzumab treatment due to primary resistance and acquired resistance (see, for example, Valabrega G, Montemurro F, Aglietta M) .Ann Oncol.2007;18(6):977–84). Pertuzumab is another humanized monoclonal IgG1 antibody that has been approved for use in HER2-positive breast cancer patients (Sandrine Richard et al, Annals of the Brazilian Academy of Sciences. 2016; 88 (1 Suppl. ): 565-577). It binds to the extracellular domain II of HER2 and blocks the dimerization of HER2 with other HER family members (especially Her3), thereby inhibiting downstream signal transduction processes associated with tumor growth and development (Adams CW et al, Cancer Immunol Immunother. 2006; 55:717–27; Franklin MC et al., Cancer Cell. 2004; 5:317–28). Since the binding site of pertuzumab is different from trastuzumab, the combination therapy of these two antibodies has been shown to enhance antitumor activity (Scheuer W et al, Cancer Res. 2009; 69(24): 9330– 6). Pertuzumab in combination with trastuzumab plus docetaxel has been approved for first-line treatment in patients with HER2-positive metastatic breast cancer (MBC) (Gideon M, Blumenthal et al, Clin Cancer Res. 2013;19(18)). However, most patients with HER2-metastatic breast cancer still relapse after treatment with currently available HER2-targeted therapies. Therefore, other treatment options are still urgently needed.

Summary of the invention

Bispecific antibodies (BsAbs) are a promising strategy to overcome resistance to currently available treatment options. Bispecific antibodies are capable of efficiently recruiting and activating immune cells and directly targeting tumor cells compared to conventional antibodies. A variety of BsAbs have been developed which redirect tumor cytotoxicity via CD3 on T cells or CD16 on NK cells. A bispecific antibody Her-S-Fab in which trastuzumab Fab is linked to an anti-CD16 single domain antibody has been previously prepared (Aifen Li et al, AMB Expr. 2016; 6:32). The Her-S-Fab can induce potent cytotoxicity in HER2-positive cells. In the present application, the present inventors prepared a similar bispecific antibody, Her2(Per)-S-Fab, using a pertuzumab Fab and an anti-CD16 single domain antibody, and unexpectedly found that Her2( Per)-S-Fab is even more cytotoxic to HER2-positive tumor cells than Her-S-Fab and thus can be used for the treatment of HER2-positive tumors.

Thus, in a first aspect, the present application relates to a multispecific antibody comprising a binding moiety that specifically binds to the extracellular domain II of HER2 and a binding moiety that specifically binds to an antigen on an immune effector cell. The antibody is preferably a bispecific antibody.

In a second aspect, the application provides a multispecific antibody conjugate comprising the multispecific antibody conjugated to a nanoparticle. The conjugate may also comprise a binding moiety that specifically binds to the HER2 extracellular domain IV.

In another embodiment, the application provides a multispecific antibody conjugate comprising a binding moiety that specifically binds to the extracellular domain II of HER2 and a binding moiety that specifically binds to an antigen on an immune effector cell, wherein The binding moiety is coupled to a nanoparticle, the binding moiety being an antibody or antibody fragment. The conjugate may further comprise a binding moiety that specifically binds to the extracellular domain IV of HER2, wherein the binding moiety that specifically binds to the extracellular domain II of HER2 and the binding moiety that specifically binds to the extracellular domain IV of HER2 may be multispecific antibodies ( As part of a bispecific antibody.

In the first and second embodiments, the binding portion that specifically binds to the extracellular domain II of HER2 may be pertuzumab or an antibody fragment from pertuzumab. The binding portion that specifically binds to the extracellular domain II of HER2 may also be an antibody or antibody fragment that competes with pertuzumab for binding to a binding site on the extracellular domain II of HER2, eg, the binding moiety is identical to the binding of pertuzumab gauge.

The binding portion that specifically binds to the extracellular domain IV of HER2 is trastuzumab or an antibody fragment from trastuzumab. The binding portion that specifically binds to the HER2 extracellular domain IV may also be an antibody or antibody fragment that competes with trastuzumab for binding to the binding site on the HER2 extracellular domain IV, eg, the binding moiety is identical to trastuzumab gauge.

In another aspect, the present application provides a pharmaceutical composition, a multispecific antibody or conjugate of the present application, which is preferably used to treat a HER2-positive tumor. The pharmaceutical composition optionally comprises other therapeutic agents, such as docetaxel.

In a further aspect, the application provides a method of treating a HER2-positive tumor, the method comprising administering an effective amount of a multispecific antibody or conjugate or pharmaceutical composition described herein.

In another aspect, the application provides the use of the multispecific antibody or conjugate described in the manufacture of a medicament for the treatment of a HER2-positive tumor.

A HER2-positive tumor that can be treated with a multispecific antibody, conjugate or pharmaceutical composition of the present application can be selected from the group consisting of breast cancer, lung cancer, ovarian cancer, gastric cancer, bladder cancer, pancreatic cancer, endometrial cancer, colon cancer, kidney. Cancer, esophageal cancer, and prostate cancer are preferably breast cancer and colon cancer.

This application relates to the following embodiments:

A multispecific antibody comprising a binding portion that specifically binds to the extracellular domain II of HER2 and a binding portion that specifically binds an antigen on an immune effector cell, wherein the immune effector cell preferably comprises T cells, NK cells, and/or NKT cells.

2. The multispecific antibody of clause 1, wherein the antibody is a bispecific antibody.

3. A multispecific antibody conjugate comprising a multispecific antibody according to

clause

1 or 2 coupled to a nanoparticle.

4. The conjugate according to item 3, which further comprises a binding moiety that specifically binds to the HER2 extracellular domain IV.

5. A multispecific antibody conjugate comprising a binding moiety that specifically binds to the extracellular domain II of HER2 and a binding moiety that specifically binds to an antigen on an immune effector cell, wherein the binding moiety is coupled to the nanoparticle The binding moiety is an antibody or antibody fragment.

6. The conjugate of clause 5, further comprising a binding moiety that specifically binds to the HER2 extracellular domain IV.

7. The conjugate according to item 5, which comprises a multispecific antibody having a binding portion that specifically binds to the extracellular domain II of HER2 and a binding portion that specifically binds to the extracellular domain IV of HER2, wherein the conjugate The specific antibody is preferably a bispecific antibody.

The multispecific antibody or conjugate according to any one of the items 1-7, wherein the binding portion that specifically binds to the extracellular domain II of HER2 binds to the same epitope as pertuzumab.

9. The multispecific antibody or conjugate according to item 8, wherein the binding portion that specifically binds to the extracellular domain II of HER2 is pertuzumab or an antibody fragment from pertuzumab, the antibody The fragment is preferably a variable region fragment or Fab from pertuzumab.

The multispecific antibody or conjugate according to any one of the items 6-9, wherein the binding portion that specifically binds to the HER2 extracellular domain IV is trastuzumab or from trastuzumab An antibody fragment, preferably a variable region fragment or Fab from trastuzumab.

The multispecific antibody or conjugate according to any one of the items 6-9, wherein the binding portion that specifically binds to the HER2 extracellular domain IV binds to the same epitope as trastuzumab.

The multispecific antibody or conjugate according to any one of the items 1-11, wherein the immune effector cells comprise T cells.

The multispecific antibody or conjugate according to any one of claims 1 to 12, wherein the immune effector cells comprise NK cells.

A pharmaceutical composition comprising the multispecific antibody or conjugate according to any one of items 1 to 13, which is preferably for use in the treatment of a HER2-positive tumor, the HER2-positive tumor preferably being selected from the group consisting of Breast cancer, lung cancer, ovarian cancer, gastric cancer, bladder cancer, pancreatic cancer, endometrial cancer, colon cancer, kidney cancer, esophageal cancer, and prostate cancer, more preferably breast cancer, colon cancer, and the pharmaceutical composition optionally includes Other therapeutic drugs, such as docetaxel.

A method of treating a HER2-positive tumor, the method comprising administering an effective amount of the multispecific antibody or conjugate or pharmaceutical composition according to any one of items 1 to 14, the HER2-positive tumor preferably being selected from the group consisting of Breast cancer, lung cancer, ovarian cancer, gastric cancer, bladder cancer, pancreatic cancer, endometrial cancer, colon cancer, renal cancer, esophageal cancer, and prostate cancer are more preferably breast cancer.

16. Use of a multispecific antibody or conjugate according to any one of clauses 1 to 13 for the preparation of a medicament for the treatment of a HER2-positive tumor, preferably selected from the group consisting of breast cancer, lung cancer, ovary Cancer, gastric cancer, bladder cancer, pancreatic cancer, endometrial cancer, colon cancer, kidney cancer, esophageal cancer, and prostate cancer, more preferably breast cancer, colon cancer, the drug optionally including other therapeutic drugs, such as docetaxel Race.

17. The conjugate according to clause 3 or 4, which further comprises an additional binding moiety that specifically binds to an antigen on an immune effector cell.

The conjugate according to any one of items 5 to 13, wherein the binding portion that specifically binds to the extracellular domain II of HER2 and the binding portion of the antigen that specifically binds to an immune effector cell are respectively coupled to the nanoparticle. on.

The multispecific antibody or conjugate according to any one of items 1 to 13 and 17 to 18, which is for use in the treatment of a HER2-positive tumor, preferably selected from the group consisting of breast cancer, lung cancer, and ovarian cancer The gastric cancer, bladder cancer, pancreatic cancer, endometrial cancer, colon cancer, kidney cancer, esophageal cancer, and prostate cancer are more preferably breast cancer or colon cancer.

The multispecific antibody, conjugate, pharmaceutical composition, method or use of any one of clauses 1 to 19, wherein the binding portion of the antigen that specifically binds to an immune effector cell is an anti-CD16 single domain The antibody or comprises an anti-CD16 single domain antibody.

The multispecific antibody, conjugate, pharmaceutical composition, method or use of claim 20, wherein the anti-CD16 single domain antibody is an anti-CD16 VHH as set forth in SEQ ID No: 1.

The multispecific antibody, conjugate, pharmaceutical composition, method or use of any one of items 1-21, wherein the immune effector cells comprise T cells and NK cells.

DRAWINGS

Figure 1. Her2(Per)-S-Fab can be purified from E. coli. a) Bacterial expression construct of Her2(Per)-S-Fab. Each construct contains a pelB signal sequence for cytoplasmic expression. Co-expression of anti-HER2-VL-CL and anti-HER2-VH-CH1-CD16 VHH resulted in the formation of Her2(Per)-S-Fab. A Flag tag or a His8 tag was added to the C-terminus of each construct to facilitate protein detection and purification. b) Schematic representation of Her2(Per)-S-Fab after co-expression. c) Coomassie blue staining of Her2(Per)-S-Fab after immobilized Ni-NTA affinity chromatography, the anti-HER2-VL-CL approaching 25 kd, and the anti-HER2-VH-CH1-CD16 VHH is close 40kd. M, molecular weight reference, unit: kD. d) Gel filtration chromatography showed that the size of Her2(Per)-S-Fab was approximately 65 kD. Upper panel: protein markers with different molecular weights; lower panel: Her2(Per)-S-Fab. X-axis: fractions; Y-axis: protein absorbance readings as determined by UV.

Figure 2. Purified Her2(Per)-S-Fab recognizes HER2 antigen on cancer cells. A-e) Flow cytometric analysis of HER2 negative cell line (CHO) and HER2 positive cell lines (SKOV3, SKBR3, LS174T and MCF7) using Her2(Per)-S-Fab and pertuzumab. Gray areas (blank) indicate unstained cells, dotted lines (control) indicate only anti-human IgG (H&L)-FITC stained cells, and dashes indicate paclizumab and anti-human IgG (H&L)- Cells of both FITCs, while the solid line (Her2(Per)-S-Fab) indicates cells with both Her2(Per)-S-Fab and anti-human IgG (H&L)-FITC. X axis: intensity; Y axis: count. f) Western blot analysis of HER2 expression in different cell lines used in the flow cytometry studies.

Figure 3. Her2(Per)-S-Fab induces NK cell mediated cytotoxicity. a) Cytotoxicity assays were performed on different cell lines as described in the Materials and Methods section. Bf) CHO(b), MCF7(c), LS174T(d), SKBR3(e) and SKOV3 as target cells in the presence of Her2(Per)-S-Fab with or without human NK cells (f) A dose-dependent cytotoxicity assay (E/T 10:1) was performed. The concentration of Her2(Per)-S-Fab is from 0.001 ng/ml to 10 μg/ml. The mixture was incubated for 72 h and then subjected to a cytotoxicity assay. All data are averages in triplicate and error bars indicate standard deviation.

Figure 4. Cytotoxicity of different concentrations of Her2(Per)-S-Fab or HER2-S-Fab against LS174T (top panel), SKBR3 (middle panel) and SKOV3 (bottom panel). Using human NK cells as effector cells, LS174T (top panel), SKBR3 (middle panel) and SKOV3 (bottom panel) were used as target cells in the presence of different concentrations of Her2(Per)-S-Fab or HER2-S-Fab. Cytotoxicity assay (E/T 10:1). All data are in triplicate and the error bars indicate standard deviation (*P<0.05, t-test, control (no antibody) relative to the indicated concentration of Her2(Per)-S-Fab or Her-S- Fab).

Figure 5. Her2(Per)-S-Fab inhibits tumor growth in vivo. A mixture of LS174T cells (1x10 ⁶ ) and freshly isolated human PBMC (5x106) was subcutaneously implanted into NOD/SCID mice (n=6/group). Mice were subsequently treated intraperitoneally with vehicle (control, solid line) or Her2(Per)-S-Fab (20 [mu]g/dose, dashed line). The data represents the mean tumor volume of 6 mice. Error bars indicate standard deviation (*P<0.05, t-test, PBS vs. Her2(Per)-S-Fab).

Figure 6 shows the results of flow cytometry analysis (FCAS) determination of Her2(Per)-S-Fab) and HER2-S-Fab. Where 1 indicates HER2-S-Fab and 2 indicates Her2(Per)-S-Fab.

detailed description

Before describing the multispecific antibodies, antibody conjugates, pharmaceutical compositions, methods, and uses of the invention, it is to be understood that the invention is not limited to the multispecific antibodies, antibody conjugates, pharmaceutical compositions, methods described. And use, so of course it may be different. It is also understood that the terminology used herein is for the purpose of description The description of the embodiments is provided to provide a person of ordinary skill in the art the complete disclosure and description of the invention, and is not intended to limit the scope of the invention to the inventor, and is not intended to indicate that the following experiments are performed. All or only experiments. Efforts have been made to ensure the accuracy of the numbers used (eg, volume, temperature, etc.), but some experimental errors and deviations should be considered.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Some potential and preferred methods and materials are now described, although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All publications mentioned herein are hereby incorporated by reference to disclose and describe the methods and/or materials relating to the cited publication. It will be understood that in the event of a conflict, any disclosure in the cited publication is superseded by the present disclosure.

As will be apparent to those skilled in the art upon reading this disclosure, each individual embodiment described and illustrated herein has discrete components and features that can be readily separated or combined with features in several other embodiments. The scope or spirit of the invention is not departed. Any of the enumerated methods can be implemented in the order of the recited events or in any other order that is logically possible.

"A" as used herein and in the appended claims means "a" or "an".

Where a range of values is provided, it is to be understood that each intermediate value (up to one tenth of the lower limit unit) between the upper and lower limits of the range is also specifically disclosed unless the context clearly dictates otherwise. The various smaller ranges and any such values or intermediate values that are within the range are included in the invention. The upper and lower limits of these smaller ranges may be independently included in or excluded from the range, and are subject to any specific exclusion of the range, wherein either or both of the upper and lower limits Each of the ranges included in the smaller range is also included in the present invention. Where the stated range encompasses one or both of the limits, the scope of the limitation of the inclusion of either or both is also included in the invention.

Throughout this disclosure, the terms "ErbB2", "ErbB2 receptor", "c-Erb-B2" and "HER2" are used interchangeably and, unless otherwise indicated, refer to a native sequence ErbB2 human polypeptide or a functional derivative thereof.

As used herein, "specifically binds" means that the binding moiety is capable of binding the antigen with sufficient affinity or binding to HER2 on the extracellular domain such that the antibody or antibody conjugate can be used as a targeted expression. A therapeutic agent for cells of HER2. In one embodiment, the binding portion that specifically binds to HER2 on the extracellular domain II or IV binds to an unrelated non-HER2 protein to a lesser extent than about 10% of the binding of the binding moiety to HER2, as for example by an enzyme-linked immunosorbent assay (ELISA) ), based on surface plasmon resonance (SPR) assays (eg, Biacore) or flow cytometry (FACS). In certain embodiments, the binding moiety or bispecific antibody that specifically binds to HER2 has < 1 μμ, < 100 nM, < 10 nM, < 1 nM, < 0.1 nM, < 0.01 nM, or < 0.001 nM (eg, 10-8 M) Or less, such as a dissociation constant (Kd) of 10-8 M to 10-13 M, such as 10-9 M to 10-13 M).

As used herein, "antibody" refers to the immunological activity (ie, specificity) of a full-length (ie, naturally occurring or formed by a normal immunoglobulin gene fragment rearrangement process) of an immunoglobulin molecule (eg, an IgG antibody) or an immunoglobulin molecule. Sexually bound) parts, such as antibody fragments. "Antibody" includes monoclonal antibodies, polyclonal antibodies, bispecific antibodies, multispecific antibodies, murine antibodies, heavy chain antibodies (such as camelid antibodies), chimeric antibodies, humanized and human antibodies, and the like. A "heavy chain antibody" is an antibody that contains only a heavy chain and does not contain a light chain, such as a camelid antibody, a cartilage fish such as a shark antibody, and the like.

An "antibody fragment" is a part of an intact antibody, such as F(ab') ₂ , F(ab) ₂ , Fab', Fab, Fv, scFv, dAb, and the like. Regardless of the structure, the antibody fragment binds to the same antigen as recognized by the full length antibody. For example, an antibody fragment comprises an isolated fragment consisting of a variable region, such as an "Fv" fragment consisting of a heavy or light chain variable region or a recombinant single sequence in which the light chain and heavy chain variable regions are joined by a peptide linker. Chain polypeptide molecule ("scFv protein"). "Single-chain antibodies" are usually abbreviated as "the scFv", the polypeptide chain comprises V _H and V _L, composed of domains, said domains interact to form an antigen binding site. V _H domains and V _L, typically linked by a peptide having 1 to 25 amino acid residues. Antibody fragments also include bifunctional antibodies, trifunctional antibodies, and single domain antibodies (dAbs). "Antibody fragments" also include fragments of heavy chain antibodies, such as single domain antibodies (sdAbs) or fragments comprising variable regions, such as VHH.

As used herein, "Fab" refers to an antibody fragment comprising a light chain fragment comprising a VL domain and a light chain constant domain (CL) and a VH domain and a heavy chain first constant domain (CH1). In the Fab, either the variable or constant regions of the heavy and light chains may be exchanged.

A "multispecific antibody" is an antibody that can simultaneously bind at least two targets having different structures (eg, two different antigens, two different epitopes on the same antigen). A "bispecific antibody" is an antibody that can simultaneously bind two targets with different structures.

"Binding the same epitope" as a reference antibody refers to blocking 50% or more of the binding of a reference antibody to its antigen in a competition assay, and conversely, the reference antibody is antigenic to the antibody in a competition assay. The combination blocks 50% or more. An exemplary competitive assay is described, for example, in CN105829347A.

An "effective amount" of an agent (eg, a multispecific antibody, conjugate, pharmaceutical composition) refers to an amount effective to achieve the desired therapeutic or prophylactic result at the necessary dosages and schedules.

Multispecific antibody

The application provides a multispecific antibody comprising a binding moiety that specifically binds to the extracellular domain II of HER2 and a binding moiety that specifically binds to an antigen on an immune effector cell. The antibody is preferably a bispecific antibody.

The binding portion that specifically binds to the extracellular domain II of HER2 may be an antibody fragment that competes with pastuzumab for binding to the HER2 extracellular domain II or to the same epitope. The binding portion that specifically binds to the extracellular domain II of HER2 may be pertuzumab or an antibody fragment from pertuzumab, preferably a variable region fragment from pertuzumab, scFv or Fab.

The immune effector cell can be a cytotoxic effector cell, such as a cytotoxic cell. The cytotoxic cell can be a white blood cell. The leukocytes may be selected from the group consisting of macrophages, neutrophils, eosinophils, NK cells, B cells, and T cells. The white blood cells can be NK cells. The leukocytes may be NK-like T cells, ie NKT cells. The white blood cells may be NK cells and T cells.

The antigen on the exemplary T cells is selected from the group consisting of CD2, CD3, CD4, CD5, CD6, CD8, CD25, CD28, CD30, CD40, CD40L, CD44, CD45, CD69 and CD90. The antigen on the NK cells may be selected from the group consisting of CD2, CD8, CD11b, CD16, CD38, CD56, CD57, ADAM17, KIR, KAR, KLR and CD137. The antigen on an exemplary monocyte is selected from the group consisting of CD74, HLA-DRα chain, CD14, CD16, CD64, and CD89. The antigen on exemplary neutrophils is selected from the group consisting of CEACAM6, CEACAM8, CD16b, CD32a, CD89, CD177, CD11a, CD11b, and SLC44A2. The antigen on the leukocytes may also be a checkpoint antigen and may be selected from the group consisting of LSECtin, CTLA4, PD1, PD-L1, LAG3, B7-H3, B7-H4, KIR and TIM3. Preferably, the antigen on the T cell is CD3, CD4 or CD8, or the antigen on the NK cell is CD16 or CD56.

The binding moiety that specifically binds to an antigen on an immune effector cell can be an antibody fragment from an antibody that binds to the antigen, such as a Fab, scFv, a single domain antibody, an antibody fragment comprising VHH, or VHH. The binding moiety that specifically binds to an antigen on an immune effector cell is preferably an antibody fragment (e.g., Fab, scFv or VHH) derived from an anti-CD16 antibody or an anti-CD3 antibody.

Multispecific antibody conjugate

The application provides a multispecific antibody conjugate comprising a multispecific antibody as described above conjugated to a nanoparticle. In one embodiment, the conjugate further comprises a binding moiety that specifically binds to the HER2 extracellular domain IV.

The present application also provides a multispecific antibody conjugate comprising a binding moiety that specifically binds to the extracellular domain II of HER2 and a binding moiety that specifically binds an antigen on an immune effector cell, wherein the binding moiety is coupled On the nanoparticles, the binding moiety is an antibody or antibody fragment. The binding portion that specifically binds to the extracellular domain II of HER2 and the binding portion that specifically binds to the antigen on the immune effector cell can be coupled to the nanoparticles, respectively. In one embodiment, the conjugate further comprises a binding moiety that specifically binds to the HER2 extracellular domain IV. The conjugate may comprise a multispecific antibody having a binding moiety that specifically binds to the extracellular domain II of HER2 and a binding moiety that specifically binds to the extracellular domain IV of HER2, wherein the multispecific antibody is preferably a bispecific antibody.

In the multispecific antibodies or conjugates of the present application, the binding portion that specifically binds to the extracellular domain II of HER2 can compete with pertuzumab for binding to the HER2 extracellular domain II or to the same epitope.

In the multispecific antibody or conjugate of the present application, the binding portion that specifically binds to the extracellular domain II of HER2 is pertuzumab or an antibody fragment from pertuzumab, preferably from antibody fragment Variable region fragment or Fab of pertuzumab.

In the multispecific antibodies or conjugates of the present application, the binding portion that specifically binds to the HER2 extracellular domain IV can compete with trastuzumab for binding to the HER2 extracellular domain IV or to the same epitope.

And X. Variable region fragment or Fab.

The nanomaterial used in the multispecific antibody conjugate of the present application may be a pharmaceutically acceptable nanomaterial, preferably a biodegradable nanomaterial, more preferably polylactic acid-glycolic acid, polylactic acid, polycaprolactone, polybutan Any one or a mixture of at least two of a glycol succinate, a polyaniline, a polycarbonate, an ethylene lactide copolymer or a glycolide caprolactone copolymer, most preferably a polylactic acid-glycolic acid ( PLGA), polylactic acid (PLA) and/or polycaprolactone (PCL). These nanomaterials and methods for their preparation are known in the art, for example, the nanomaterials can be prepared using the methods described below.

PCT/CN2018/073785 describes antibody conjugates comprising a multispecific antibody conjugated to a nanomaterial, such as a bispecific antibody (the content of which is incorporated by reference) The full text is introduced in this article). According to the description in the specification of the patent application, after multi-specific antibodies (such as bispecific antibodies) are coupled to the nanomaterial, the technical effects and even the technical effects of the multispecific antibodies (such as bispecific antibodies) can still be achieved. There is also improvement. For example, according to the description of the PCT application, a bispecific antibody (CD16-MUC1 BiTE) comprising anti-CD16 VHH and anti-MUCl VHH or a bispecific antibody (CD3-CEA BiTE) comprising anti-CD3 Fab and anti-CEA VHH When combined with PLGA nanoparticles, the combined killing rate of tumor cells with NK cells or T cells is higher than that of the corresponding bispecific antibody (CD16-MUC1 BiTE or CD3-CEA BiTE) combined with NK cells or T cells. rate. Thus, one skilled in the art can reasonably foresee that a similar technical effect can be achieved by coupling a multispecific antibody (such as a bispecific antibody) described herein to a nanomaterial.

According to the description of WO2016/165632 and WO2017/185662, bispecific binding conjugates prepared by coupling two antibodies or antigen-binding portions thereof for different antigens to nanoparticles, respectively, can also be like ordinary bispecific Anti-tumor effects are achieved by antibodies (ie, bispecific antibodies that are not conjugated to nanoparticles). For example, a bispecific binding conjugate (anti-CD3-PLGA-anti-her2) prepared by coupling an anti-CD3 antibody and an anti-her2 antibody to PLGA nanoparticles, combined with T cells, can kill cancer cells MCF-7. Up to 63.84% (see WO2016/165632, eg Table 5); antibodies that specifically bind NK cells (eg anti-CD16 antibodies, anti-CD56 antibodies) and antibodies that specifically bind to tumor cells (eg anti-MUC1 antibodies, anti-CD19 antibodies, Anti-CD20 antibody) bispecific binding conjugates (such as anti-CD16-PLGA-anti-MUC1, anti-CD56-PCL-anti-CD19, etc.) prepared by coupling to nanoparticles, combined with NK cells to kill tumor cells Up to 80% or more (see WO2017/185662), including a multispecific binding conjugate that specifically binds to the binding portion of T cells and a binding portion that specifically binds to NK cells (eg CD3/CD16-PLGA-MUC1 trispecific) The binding rate of the binding conjugate to the tumor cells is relatively higher. Thus, one skilled in the art can reasonably foresee that each antigen binding portion of a multispecific antibody (such as a bispecific antibody) described herein (specifically binds to the binding portion of HER2 extracellular domain II and specific binding immune effect) A similar technical effect can also be achieved by multi-specific (e.g., bispecific) binding conjugates prepared by coupling the antigen on the cell to the nanomaterial.

Preparation of antibody conjugates of the present application

The multispecific antibody of the present application is described below by taking a multispecific antibody conjugate comprising a binding moiety coupled to a nanoparticle (in this application, "nanoparticle" and "nanoparticle" are used interchangeably) as an example. A method of preparing a conjugate. The preparation method comprises the following steps:

(1) Preparation, collection and activation of nanomaterials;

(2) The nanomaterial obtained in the step (1) is bonded to the binding portion or a mixture thereof.

In the step (1), the preparation of the nano material comprises: completely dissolving the nano material with a solvent, stirring, adding water to form a uniform emulsion. Wherein the agitation may be carried out at a rotational speed of 500 to 20,000 rpm/min, for example, the rotational speed may be 500 rpm/min, 700 rpm/min, 800 rpm/min, 1000 rpm/min, 1100 rpm/min, 1200 rpm/min, 1300 rpm/min, 1400 rpm/ Min, 1480 rpm/min, 1500 rpm/min, 2000 rpm/min, 2200 rpm/min, 2500 rpm/min, 3000 rpm/min, 3500 rpm/min, 4000 rpm/min, 4200 rpm/min, 4500 rpm/min, 5000 rpm/min, 5500 rpm/min, 6000 rpm/min, 6500 rpm/min, 7000 rpm/min, 7500 rpm/min, 8000 rpm/min, 8500 rpm/min, 9000 rpm/min, 9500 rpm/min, 10000 rpm/min, 11000 rpm/min, 12000 rpm/min, 13000 rpm/min, 14000 rpm/ Min, 15000 rpm/min, 16000 rpm/min, 17000 rpm/min, 18000 rpm/min, 19000 rpm/min or 20000 rpm/min. Higher speeds can be used if necessary.

Preferably, the nano material is polylactic acid-glycolic acid (PLGA), polylactic acid (PLA), polycaprolactone (PCL), polytetramethylene glycol succinate, polyaniline, polycarbonate, ethylene-propylene cross Any one or a mixture of at least two of an ester copolymer or a glycolide caprolactone copolymer.

Preferably, the solvent is any one of acetone, methyl ethyl ketone, methanol, ethanol or isopropyl alcohol or a mixture of at least two.

Preferably, the collecting of the nanomaterial comprises: collecting the prepared nanomaterial by centrifugation, then resuspending it with deionized water, and repeating the operation of washing the nanomaterial twice. The centrifugation can be carried out at a rotational speed of 8000-15000 rpm/min, for example, the rotational speed can be 8000 rpm/min, 9000 rpm/min, 10000 rpm/min, 11000 rpm/min, 12000 rpm/min, 13000 rpm/min, 14000 rpm/min, 14500 rpm/min. , 14800 rpm / min, 15000 rpm / min. Higher speeds can be used if necessary. Nanomaterials (nanoparticles) can be collected or further purified by other methods. The nanoparticles may have an average particle diameter as described above

Preferably, the activation of the nanomaterial comprises: using 1-10 mg/mL of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDS) and N-hydroxysuccinyl The amine (NHS) mixed solvent activates the nanomaterial at room temperature for 0.5-5 h.

In step (2), the joining comprises: collecting the activated nanomaterial by centrifugation, and then washing the nanomaterial once with the ligation reaction solution. Adding a binding moiety to be joined, or an equal mixture of two or more binding moieties to the ligation reaction solution, and then resuspending the nanomaterial with a ligation reaction solution containing the binding moiety or mixture, and performing a reaction at room temperature 0.5 -5h. After the reaction, the nanomaterials were collected by centrifugation, and the nanomaterials were washed twice with Dunsen's phosphate buffer solution, and then resuspended in Dun's phosphate buffer solution (D-PBS) and stored at 4 ° C until use. Other methods of activation of the nanomaterial can be employed.

The preparation method of the multispecific antibody conjugate of the present application specifically includes the following steps:

(1) Preparation of nanomaterials: completely dissolve the nanomaterials to a concentration of 5-30 mg/mL with acetone, and at a volume ratio of acetone to deionized water of 1:4, magnetically stirred at 500-1500 rpm/min. A solution of the material and acetone is added to the deionized water to form a uniform emulsion, and then stirring is continued until the acetone is volatilized;

(2) Collection of nanomaterials: The prepared nanomaterials were collected by centrifugation at 8000-15000 rpm/min, then resuspended in deionized water, and the nanomaterials were washed twice in repeated operations;

(3) Activation of nanomaterials: activation of nanometers at room temperature using 1-10 mg/mL of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide mixed solvent Material 0.5-5h;

(4) Nanomaterial-linked antibody: The activated nanomaterial is collected by centrifugation, and then the nanomaterial is washed once with 0.1 M, pH=8.0 Duns' phosphate buffer solution, the binding portion to be connected, or two or two An equal amount of the above combined portion is added to the ligation reaction solution, and then the nanomaterial is resuspended with a ligation reaction solution containing the binding moiety or mixture, and the reaction is allowed to react at room temperature for 0.5-5 h. After the reaction is completed, the nanomaterial is collected by centrifugation using Duchensenate. The nanomaterial was washed twice with a buffer solution, and then resuspended in a Dun's phosphate buffer solution and stored at 4 ° C until use.

Pharmaceutical composition, treatment method, use

The application also provides a pharmaceutical composition comprising a multispecific antibody or conjugate as described herein.

The pharmaceutical composition can be used to treat HER2-positive tumors. The HER2-positive tumor may be selected from the group consisting of breast cancer, lung cancer, ovarian cancer, gastric cancer, bladder cancer, pancreatic cancer, endometrial cancer, colon cancer, kidney cancer, esophageal cancer, and prostate cancer, preferably breast cancer, colon cancer.

The pharmaceutical composition optionally comprises other therapeutic agents. The therapeutic agent can be a cytotoxic drug such as docetaxel.

The application also provides a method of treating a HER2-positive tumor, the method comprising administering an effective amount of a multispecific antibody or conjugate or pharmaceutical composition described herein.

The application also provides the use of the multispecific antibodies or conjugates described in the manufacture of a medicament for the treatment of HER2-positive tumors.

The HER2-positive tumor and the therapeutic agent are as described above.

Example

Materials and Method

Plasmid and protein purification

The structure of Her2(Per)-S-Fab is shown in Figure 1a. VH-CH1 and VL-CL of pertuzumab were synthesized and cloned using standard DNA cloning techniques (Aifen Li, Jieyu Xing, Li Li. et al. AMB Expr. 2016; 6:32). A signal sequence pelB for periplasmic expression was added at the N-terminus (Spiess C et al, Nat Biotechnol. 2013; 31(8): 753-8). The sequence of the CD16 single domain antibody portion is shown in SEQ ID No: 1 (see Aifen Li, Jieyu Xing, Li Li. et al. AMB Expr. 2016; 6:32 cited in the literature: Behar G et al. Isolation And characterization of anti-FcgammaRIII (CD16) llama single-domain antibodies that activate natural killer cells. Protein Eng Des Sel. 2008; 21(1): 1–10). Her2(Per)-S-Fab was formed by heterodimerization of VL-CL/VH-CH1-anti-CD16 VHH (Fig. 1b). Flag-tag or His-tag was added at the C-terminus for easy detection.

For purification of Her2(Per)-S-Fab, cytoplasmic protein purification was performed as described in the previous literature (Kwong KY, Rader C. Curr Protoc Protein Sci. 2009; 6: 1-14). Briefly, two plasmids encoding a single polypeptide were co-transformed into BL21 (DE3) and grown in 2YT medium containing appropriate antibiotics (100 μg/ml kanamycin and 100 μg/ml ampicillin) at 37 °C. When the cell culture reached an OD600 of about 0.8-1.0, 0.2 mM isopropyl-β-D-thiogalactopyranoside (IPTG) was added to induce protein expression. The cells were incubated for an additional 40 hours at 16 ° C and harvested.

The cell pellet was resuspended 1:4 in a cold sucrose solution [20 mM Tris-HCl pH 8.0; 25% (w/v) sucrose; 1 mM EDTA] for cytoplasmic extraction. The suspension was centrifuged at 8500 g for 20 minutes, and the supernatant was collected as a sucrose fraction. The pellet was resuspended in a cold cytosolic solution (5 mM MgCl ₂ ; 0.15 mg/ml lysozyme; 1 mM PMSF) and centrifuged at 8500 g for 20 minutes. The supernatant was collected as a periplasmic fraction. Then, Her2(Per)-S-Fab was purified from the combined sucrose fraction and cytoplasmic fraction by immobilized Ni-NTA affinity chromatography.

Gel filtration was performed with GE Superdex 200 increase 10/300 according to the manufacturer's instructions. The gel filter protein marker was from Sigma (MWGF200).

Cells and animals

All cell lines (including: HER2 positive cell lines - SKBR3 and MCF7 (human breast cancer cells), SKOV3 (human ovarian cancer cells), LS174T (human colorectal cancer); HER2 negative cell line - Chinese hamster ovary cells (CHO) )) are purchased from the Type Culture Collection of the Chinese Academy of Sciences, Shanghai, China. SKBR3, SKOV3 and MCF7 in Dulbecco's Modified Eagle Medium (DMEM, Gibco, Life Technologies, China) containing 10% HI-fetal calf serum (Gibco, Life Technologies, USA) and 1% penicillin/streptomycin (HyClone) Cultivate. LS174T and CHO in RPMI-1640 medium (Gibco, Life Technologies, China) containing 10% HI-fetal calf serum (Gibco, Life Technologies, USA) and 1% penicillin/streptomycin (HyClone) at 37 °C Cultured in a 5% CO ₂ humidified incubator.

Non-obese diabetic-severe combined immunodeficiency disease (NOD/SCID) mice (female, 18-22 g) were purchased from Vital River Laboratory Animal Technology Co. Ltd and then under sterile standardized environmental conditions at the Animal Experimental Center of Sun Yat-sen University (20 -26 ° C room temperature, 40-70% relative humidity and 12h light and dark rhythm).

To isolate natural killer (NK) cells, peripheral blood mononuclear cells (PBMC) were prepared from freshly contributed healthy volunteer blood using Ficoll density centrifugation (Li L et al, J Immunother. 2015; 38(9): 350–6 .). Human NK cells with EasySep ^TM Enrichment Kit ^{(EasySep TM human NK cell enrichment Kit} , STEMCELL Technologies, Inc., Vancouver, Canada), was prepared from the PBMC NK cells. Prior to the cytotoxicity assay, the isolated NK cells were cultured in RPMI 1640 containing 10% FBS and 1% penicillin/streptomycin in a 37 ° C, 5% CO ₂ humidified incubator.

Flow Cytometry Analysis (FCAS)

For flow cytometry analysis, cells were harvested when cells reached 80-90% confluence. The cells were then washed twice with ice-cold phosphate buffered saline (PBS) + 0.5% bovine serum albumin (BSA). The cells were then resuspended in 100 μl of ice-cold PBS + 0.5% BSA (1 x 10 ⁶ cells). Anti-HER2 antibody trastuzumab (a gift from Alphamab, Suzou) or Her2(Per)-S-Fab was added to a final concentration of 50 μg/mL. Cells with primary antibodies were incubated on ice for 1 h and then washed three times with ice-cold PBS + 0.5% BSA. Goat anti-human IgG (H+L) Alexa Fluor 488 (Invitrogen, cat #A11013) was then added to a final concentration of 10 μg/mL. The cells were incubated for an additional hour on ice. Flow cytometry analysis was performed after washing the cells twice.

Cytotoxicity assay

Cytotoxicity assays were performed as previously described (Li L et al, J Immunother. 2015; 38(9): 350-6). Briefly, SKBR3, SKOV3, LS174T, MCF7 and CHO cells were used as target (T) cells. Isolated unstimulated NK cells were used as effector (E) cells. 100 μl of target cells (2500 cells/well) were plated in triplicate in 96-well plates. After 8 h of incubation, an equal volume of NK cells was added to each well at an E:T ratio of 10:1. The indicated concentrations of antibody are then added (ranging from 0.01 ng/ml to 10 [mu]g/ml). After 72 h of incubation, cell viability was quantified using CCK8 reagent (Dojindo, CK04) according to the manufacturer's instructions. The target cell survival rate was calculated by the following formula: [(live target cell (sample) - medium) / live target cell (control) - medium] x 100%.

In vivo tumor growth inhibition assay

In vivo tumor growth inhibition assays were performed according to the previously described methods modified (Li L et al, J Immunother. 2015; 38(9): 350-6; Junttila TT et al, Cancer Res. 2014; 74(19): 5561-71 ). Briefly, LS174T human colon cancer cells were harvested from cell culture, washed once with PBS, and then mixed with PBMC freshly prepared from healthy donors. Mixed 1×10 ⁶ LS174T cells and 5×10 ⁶ human PBMC were subcutaneously injected to the right side of NOD/SCID mice in a total volume of 0.2 mL/mouse. One hour after implantation, antibody (1 mg/kg) or vehicle control (PBS) was administered intraperitoneally. Animals were treated daily (20 μg/mouse) for the next 6 days. Tumor volume was measured in two vertical dimensions using a caliper and calculated using the formula (width ² × length)/2. All data shown are the arithmetic mean of each group.

Example 1. Her2(Per)-S-Fab can be purified from E. coli

To construct Her2(Per)-S-Fab, an anti-CD16 single domain antibody was genetically linked to the C-terminus of pertuzumab VH-CH1 (Behar G et al, Protein Eng Des Sel. 2008; 21(1): 1–10). After co-expression of VH-CH1-CD16VHH and pertuzumab VL-CL, Her2(Per)-S-Fab was formed by heterodimerization of VH-CH1-CD16 VHH with VL-CL polypeptide (Fig. 1a, 1b). The ligation of anti-CD16 single domain antibodies does not affect the expression level and solubility of the anti-HER2 Fab, probably because the anti-CD16 single domain antibody is relatively small and soluble. Her2(Per)-S-Fab was purified from the cytosol by affinity purification of Ni-NTA-agarose (Fig. 1c). The expression level of Her2(Per)-S-Fab is about 0.2 mg/L to 0.3 mg/L.

To determine if Her2(Per)-S-Fab was correctly formed as a heterodimer, gel filtration was performed to analyze the purified Her2(Per)-S-Fab. The purified Her2(Per)-S-Fab has an electrophoretic molecular weight of about 65 kD (Fig. 1d), which corresponds to the expected molecular weight of Her2(Per)-S-Fab, suggesting that most Her2(Per)-S-Fab are correct. The ground is folded into a heterodimer.

Example 2. Her2(Per)-S-Fab recognizes HER2 positive cells

To investigate whether Her2(Per)-S-Fab binds to HER2-positive cells, flow cytometry analysis was performed using HER2 positive cell lines SKBR3, SKOV3, LS174T and MCF7 and the HER2 negative cell line CHO as a control. For the HER2 negative cell line CHO, no pertuzumab or Her2(Per)-S-Fab binding was observed (Fig. 2a). The positive control pertuzumab can bind to HER2 expressing cells SKBR3, SKOV3, LS174T and MCF7 (Fig. 2b-e) (Aifen Li et al, AMB Expr. 2016; 6:32). Her2(Per)-S-Fab also binds to HER2 positive cells SKBR3, SKOV3, LS174T and MCF7 (Fig. 2b-e). Compared to low HER2 expressing cells MCF7 and low to medium HER2 expressing cells LS174T (Aifen Li et al, AMB Expr. 2016; 6:32; Vladimir Tolmachev et al, European Journal of Nuclear Medicine and Molecular Imaging. 2011; 38(3): 531–539; DWRusnak et al, Cell Proliferation. 2007; 40(4): 580-594), high HER2 expressing cells SKBR3 and SKOV3 are shown for both pertuzumab and Her2(Per)-S-Fab Higher HER2 binding (Fig. 2b-e). Her2(Per)-S-Fab showed a slightly reduced binding density compared to pertuzumab, which may be due to monovalent binding (Fig. 2b-e). Similar results based on Western blotting were also observed (Fig. 2f), with SKBR3 and SKOV3 having higher HER2 expression levels, while LS174T and MCF7 have lower HER2 expression levels. These data suggest that Her2(Per)-S-Fab can specifically bind to HER2 positive cells.

Example 3. Her2(Per)-S-Fab induces NK cell mediated cytotoxicity

In order to determine whether Her2(Per)-S-Fab can engage NK cells to kill tumor cells, an in vitro cytotoxicity assay was performed (Fig. 3). When Her2(Per)-S-Fab was incubated with tumor cells at 10 ng/ml or 100 ng/ml in the absence of NK cells, no cell killing of HER2 positive cell line SKOV3 and SKBR3 or HER2 negative cell line CHO was observed. (Figure 3a). NK cells alone did not have cytotoxic activity against all three cell lines (Fig. 3a).

However, when Her2(Per)-S-Fab was incubated with freshly isolated NK cells at a ratio of 10:1 (NK cells: tumor cells), it was present at 10 ng/ml or 100 ng/ml Her2(Per)-S-Fab. Strong cytotoxic activity against HER2-positive SKBR3 and SKOV3 cells was observed (Fig. 3a). In the presence of NK cells, no cell killing of HER2 negative cell line CHO by Her2(Per)-S-Fab was observed (Fig. 3a), indicating that Her2(Per)-S-Fab specifically triggers in the presence of NK cells. Cytotoxicity against HER2-positive tumor cells.

To further assess the cytotoxicity of Her2(Per)-S-Fab on tumor cells, a dose response to cancer cells was measured. For HER2-negative CHO cells, Her2(Per)-S-Fab showed no cytotoxicity in the presence of NK cells. In the absence of NK cells, Her2(Per)-S-Fab did not inhibit tumor cell growth in Her positive cells (Fig. 3c-f). In the presence of NK cells, Her2(Per)-S-Fab induced strong cytotoxicity against SKOV3 and SKBR3 in a dose-dependent manner (Fig. 3e, f). At the same time, much lower cytotoxicity was observed for HER2 low expressing MCF7 cells and low to moderately expressing LS174T cells (Fig. 3c, d), which further suggests that the cytotoxic activity of Her2(Per)-S-Fab is dependent on tumor cells. HER2 expression levels (Fig. 3f).

Example 4. Her2(Per)-S-Fab is more cytotoxic than HER2-S-Fab

A bispecific antibody Her-S-Fab having a structure similar to Her2(Per)-S-Fab has been previously prepared (Aifen Li et al, AMB Expr. 2016; 6:32). Her-S-Fab is constructed by linking trastuzumab Fab to anti-CD16 VHH. The bispecific antibody Her-S-Fab can induce potent cytotoxicity in HER2-positive cells. To compare the cytotoxic activities of HER2-S-Fab and Her2(Per)-S-Fab, SKOV3, SKBR3 and LS174T cells were used (Fig. 4).

In the absence of NK cells, Her2(Per)-S-Fab or Her-S-Fab had the least effect on tumor cell growth in vitro, except that Her-S-Fab alone expressed high expression of SKOV3 to HER2 at a high concentration of 1000 ng/ml. The cells showed slightly increased cytotoxicity (Figure 4, bottom panel). The Her2(Per)-S-Fab bispecific antibody exhibited higher cytotoxic activity than Her-S-Fab in the presence of NK cells (Fig. 4). Her2(Per)-S-Fab significantly inhibited cell growth at 10 ng/ml, 100 ng/ml and 1000 ng/ml, and dose-responded to all three cell lines (Fig. 4). However, for Her-S-Fab, cell growth of HER2 high expression SKOV3 and SKBR3 cells was significantly inhibited only at high concentrations of 100 ng/ml and 1000 ng/ml (Fig. 4). Low doses of 10 ng/ml of Her-S-Fab did not inhibit cell growth even for HER2 high expression of SKOV3 and SKBR3 cells (Fig. 4). In the HER2 low to moderate expression cell line LS174T, Her-S-Fab inhibited cell growth only at a high concentration of 1000 ng/ml. These data suggest that Her2(Per)-S-Fab shows higher cytotoxicity than HER2-S-Fab.

Example 5. Her2(Per)-S-Fab inhibits tumor growth in vivo

In order to examine whether Her2(Per)-S-Fab can inhibit tumor growth in vivo, an adoptive transfer model was implemented. Low to medium HER2 expressing cells LS174T cells were mixed with freshly prepared human PBMC and then subcutaneously implanted into mice. In order to minimize the effect of mouse immune cells on the implanted human cells, immunodeficient NOD/SCID mice were used in this example. After implantation, mice were treated daily with vehicle control PBS or Her2(Per)-S-Fab for 7 days. Significant anti-tumor activity was observed using 20 μg/dose of Her2(Per)-S-Fab (Fig. 5), suggesting that Her2(Per)-S-Fab can inhibit HER2 tumor growth in vivo.

discuss

In the present application, the inventors expressed and purified the HER2 targeting bispecific antibody Her2(Per)-S-Fab from E. coli. Due to the CD16 single domain binding portion and the HER2 binding portion from the pertuzumab Fab, Her2(Per)-S-Fab can recruit NK cells to effectively kill HER2 positive tumor cells. The cytotoxicity level of Her2(Per)-S-Fab correlates with the HER2 expression level of cancer cells. For example, the high HER2 expressing cell lines SKOV3 and KBR are more sensitive to Her2(Per)-S-Fab than the low to medium HER2 expressing cell line LS174T and the low HER2 expressing cell line MCF-7 (Figures 2, 3 and 4). ). Mouse xenograft studies also suggest that Her2(Per)-S-Fab inhibits tumor growth in vivo.

In recent clinical trials, immunotherapy has shown promising results as an important cancer treatment. The recruitment of immune cells (especially T cells or NK cells) to eliminate tumor cell bispecific antibodies has received great attention as a powerful cancer immunotherapy strategy. A wide variety of bispecific antibody formats ranging from IgG-like molecules to small recombinant forms have been proposed, such as tandem single-chain variable fragments (scFv) and diabodies (Db), and potential antibodies against these antibodies have been investigated. Tumor effect.

Blinatumomab (Blinatumomab) is a bispecific T cell adaptor (BiTE) format that targets CD19 on acute lymphoblastic leukemia (ALL) cells and has been approved by the FDA for the treatment of patients with B cell leukemia (Hannah Byrne et al, Trends in Biotechnology. November 2013, Vol. 31, No. 11; Oak E et al, Drugs. 2015; 24: 715-724). However, most bispecific antibody formats have problems with unpredictable expression yields and spontaneous aggregation. Compared to single-chain Fv, single domain antibodies (sdAbs), called VHHs in camels, have better biophysical properties, including less aggregation than scFv, high expression levels in E. coli, and stability in vitro. Sex and solubility are high (Hannah Byrne et al, Trends in Biotechnology. November 2013, Vol. 31, No. 11; Rozan C et al, Mol Cancer Ther. 2013; 12: 1481 - 1491). These properties provide advantages for constructing VHH-based bispecific antibodies. Therefore, the inventors designed a bispecific antibody based on the application of a single domain antibody, including Her2(Per)-S-Fab in the present application. These antibodies can be efficiently expressed in bacteria and have strong tumor killing activity in vitro and in vivo.

Another advantage of Her2(Per)-S-Fab and other single domain based bispecific antibodies is the small size. This can increase tumor penetration and achieve higher efficacy. At the same time, Her2(Per)-S-Fab exhibits greater cytotoxicity in vitro than the bispecific antibody HER2-S-Fab based on trastuzumab, which provides additional resistance to overcoming resistance to tresotuzumab. A HER2-targeted therapy.

Her2(Per)-S-Fab exhibits greater cytotoxicity than the bispecific antibody HER2-S-Fab based on trastuzumab in vitro, and cannot be based on Her2(Per)-S-Fab and HER2-S- The binding affinity of Fab to HER-positive tumor cells (their binding affinities are similar, Figure 6) is foreseen. Although not wishing to be bound by theory, it is believed that Her2(Per)-S-Fab is more cytotoxic because it binds to a different site on HER2, ie, binds to the extracellular domain II of HER2, and therefore is considered to contain specificity. A bispecific/multispecific antibody or antibody conjugate that binds to a binding portion of HER2 extracellular domain II and a binding portion that specifically binds to an antigen on an immune effector cell, compared to a binding moiety and specificity comprising a specific binding to the HER2 extracellular domain IV Bispecific/multispecific antibodies or antibody conjugates that bind to the binding portion of an antigen on an immune effector cell are more cytotoxic to HER2-negative tumor cells.

In conclusion, the novel bispecific antibody Her2(Per)-S-Fab can be efficiently expressed in E. coli and is easy to purify. It redirects NK cells to HER2-positive tumor cells and shows strong anti-tumor activity both in vitro and in vivo. Such antibodies may provide another HER2-targeted therapy for treating HER2-positive tumors and overcoming resistance to trastuzumab.

* * *

In accordance with the present disclosure, all of the conjugates, compositions, and methods disclosed and claimed herein can be prepared and used without undue experimentation. Although the compositions and methods have been described in terms of the preferred embodiments, it will be apparent to those skilled in the art that the conjugates, compositions and methods described herein can be described without departing from the spirit, scope and scope of the invention. And the steps or steps in the method are applied in a sequence. More specifically, certain reagents that are chemically and physiologically related may be substituted for the reagents described herein while achieving the same or similar results. All such similar substitutes and modifications are obvious to those skilled in the art, and are within the spirit, scope and concept of the invention as defined by the appended claims.

Claims

A multispecific antibody comprising a binding portion that specifically binds to the extracellular domain II of HER2 and a binding portion that specifically binds to an antigen on an immune effector cell, wherein the immune effector cell preferably comprises a T cell, an NK cell, and/or an NKT cell The antibody is preferably a bispecific antibody.
A multispecific antibody conjugate comprising the multispecific antibody of claim 1 conjugated to a nanoparticle.
The conjugate according to claim 2, further comprising a binding moiety that specifically binds to the HER2 extracellular domain IV.
a multispecific antibody conjugate comprising a binding moiety that specifically binds to the extracellular domain II of HER2 and a binding moiety that specifically binds to an antigen on an immune effector cell, wherein the binding moiety is coupled to the nanoparticle, The binding moiety is an antibody or antibody fragment.
The conjugate according to claim 4, further comprising a binding moiety that specifically binds to the HER2 extracellular domain IV.
The conjugate according to claim 5, comprising a multispecific antibody having a binding portion that specifically binds to the extracellular domain II of HER2 and a binding portion that specifically binds to the extracellular domain IV of HER2, wherein said multispecific The sex antibody is preferably a bispecific antibody.
The multispecific antibody or conjugate according to any one of claims 1 to 6, wherein the binding portion that specifically binds to the extracellular domain II of HER2 is pertuzumab or an antibody from pertuzumab a fragment; or the binding portion that specifically binds to the extracellular domain II of HER2 binds to the same epitope as pertuzumab.
The multispecific antibody or conjugate according to any one of claims 6 to 7, wherein the binding portion that specifically binds to the extracellular domain IV of HER2 is trastuzumab or an antibody derived from trastuzumab The fragment; or the binding portion that specifically binds to the extracellular domain IV of HER2 binds to the same epitope as trastuzumab.
A pharmaceutical composition comprising the multispecific antibody or conjugate according to any one of claims 1-8, preferably for treating a HER2-positive tumor, the HER2-positive tumor preferably being selected from the group consisting of breast Cancer, lung cancer, ovarian cancer, gastric cancer, bladder cancer, pancreatic cancer, endometrial cancer, colon cancer, kidney cancer, esophageal cancer, and prostate cancer, more preferably breast cancer, colon cancer, and the pharmaceutical composition optionally includes other Therapeutic drugs, such as docetaxel.
The use of a multispecific antibody or conjugate according to any one of claims 1-8 for the preparation of a medicament for the treatment of a HER2-positive tumor, preferably selected from the group consisting of breast cancer, lung cancer, ovarian cancer , gastric cancer, bladder cancer, pancreatic cancer, endometrial cancer, colon cancer, kidney cancer, esophageal cancer, and prostate cancer, more preferably breast cancer, colon cancer, the drug optionally including other therapeutic drugs, such as docetaxel .