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Msg  10134 of 10312  at  8/11/2022 1:07:31 AM  by

JBWIN


Building IP: BMY Patent Application re "ANTI-CTLA4 ANTIBODY PRODRUGGABLE (PROBODY) AT A CDR POSITION"

 
United States Patent Application20220251206
Kind CodeA1
KRYSTEK JR.; Stanley R. ; et al.August 11, 2022

ANTI-CTLA4 ANTIBODY PRODRUGGABLE (PROBODY) AT A CDR POSITION

Abstract

An anti-CTLA4 antibody can be prodrugged by replacement of certain complementarity determining region (CDR) amino acids with a cysteine (Cys), which provides a chemical functionality for attachment of a blocking moiety that can be cleaved at the site of intended action, thus releasing the un-prodrugged antibody.


Inventors:KRYSTEK JR.; Stanley R.; (Ringoes, NJ) ; RAO-NAIK; Chetana; (Walnut Creek, CA) ; VITE; Gregory D.; (Titusville, NJ) ; MORIN; Paul E.; (Pennington, NJ) ; LIN; Zheng; (North Wales, PA)
Applicant:
NameCityStateCountryType

BRISTOL-MYERS SQUIBB COMPANY

Princeton

NJ

US
Family ID:1000006333924
Appl. No.:17/617552
Filed:June 8, 2020
PCT Filed:June 8, 2020
PCT NO:PCT/US2020/036574
371 Date:December 8, 2021

Related U.S. Patent Documents

Application NumberFiling DatePatent Number
62859835Jun 11, 2019

Current U.S. Class:1/1
Current CPC Class:A61P 35/00 20180101; C07K 16/2818 20130101; A61K 2039/505 20130101; C07K 2317/515 20130101; C07K 2317/24 20130101
International Class:C07K 16/28 20060101 C07K016/28; A61P 35/00 20060101 A61P035/00

Claims



1. An anti-CTLA4 antibody having (i) a heavy chain CDR1 comprising the sequence of amino acids of SEQ ID NO:1, (ii) a heavy chain CDR2 comprising the sequence of amino acids of SEQ ID NO:2, (iii) a heavy chain CDR3 comprising the sequence of amino acids of SEQ ID NO:3, (iv) a light chain CDR1 comprising the sequence of amino acids of SEQ ID NO:4, (v) a light chain CDR2 comprising the sequence of amino acids of SEQ ID NO:5, and (iv) a light chain CDR3 comprising the sequence of amino acids of SEQ ID NO:6; wherein one of Xaa at position 8 of SEQ ID NO:.sub.2, at position 5 of SEQ ID NO:4, at position 8 of SEQ ID NO:4, at position 3 of SEQ ID NO:5, or at position 7 of SEQ ID NO:5 is Cys.

2. An antibody according to claim 1, wherein Xaa at position 8 of SEQ ID NO:2 is Cys.

3. An antibody according to claim 1, wherein Xaa at position 5 of SEQ ID NO:4 is Cys.

4. An antibody according to claim 1, wherein Xaa at position 8 of SEQ ID NO:4 is Cys.

5. An antibody according to claim 1, wherein Xaa at position 3 of SEQ ID NO:5 is Cys.

6. An antibody according to claim 1, wherein Xaa at position 7 of SEQ ID NO:5 is Cys.

7. A prodrugged antibody according to formula (I) (BM-L).sub.m-Ab (I) wherein Ab is an antibody having (i) a heavy chain CDR1 comprising the sequence of amino acids of SEQ ID NO:1, (ii) a heavy chain CDR2 comprising the sequence of amino acids of SEQ ID NO:2, (iii) a heavy chain CDR3 comprising the sequence of amino acids of SEQ ID NO:3, (iv) a light chain CDR1 comprising the sequence of amino acids of SEQ ID NO:4, (v) a light chain CDR2 comprising the sequence of amino acids of SEQ ID NO:5, and (iv) a light chain CDR3 comprising the sequence of amino acids of SEQ ID NO:6; wherein one of Xaa at position 8 of SEQ ID NO:.sub.2, at position 5 of SEQ ID NO:4, at position 8 of SEQ ID NO:4, at position 3 of SEQ ID NO:5, or at position 7 of SEQ ID NO:5 is Cys; BM is a blocking moiety that inhibits binding of Ab to its antigen; each L is, independently, a linker moiety bonded to BM and Ab, L comprising a cleavable moiety and being bonded to Ab at a Cys at position 8 of SEQ ID NO:.sub.2, at position 5 of SEQ ID NO:4, at position 8 of SEQ ID NO:4, at position 3 of SEQ ID NO:5, or at position 7 of SEQ ID NO:5; and m is 1 or 2.

8. A prodrugged antibody according to claim 7, wherein, in antibody Ab, Xaa at position 8 of SEQ ID NO:2 is Cys.

9. A prodrugged antibody according to claim 7, wherein, in antibody Ab, Xaa at position 5 of SEQ ID NO:4 is Cys.

10. A prodrugged antibody according to claim 7, wherein, in antibody Ab, Xaa at position 8 of SEQ ID NO:4 is Cys.

11. A prodrugged antibody according to claim 7, wherein, in antibody Ab, Xaa at position 3 of SEQ ID NO:5 is Cys.

12. A prodrugged antibody according to claim 7, wherein, in antibody Ab, Xaa at position 8 of SEQ ID NO:5 is Cys.

13. A prodrugged antibody according to claim 7, wherein blocking moiety BM is a poly(ethylene glycol).

14. A prodrugged antibody according to claim 13, wherein the poly(ethylene glycol) has a molecular weight of at least about 2 kDa.

15. A prodrugged antibody according to claim 7, wherein cleavable moiety in L is an enzymatically cleavable peptide.

16. A prodrugged antibody according to claim 15, wherein the enzymatically cleavable peptide is cleavable by at least one enzyme selected from the group consisting of fibroblast activation protein (FAP), urokinase-type plasminogen activator (uPA, urokinase), MT-SP1/matriptase, legumain, and a matrix metalloprotease.

17. A prodrugged antibody according to claim 16, wherein the enzyme is matriptase.

18. A prodrugged antibody according to claim 15, wherein the enzymatically cleavable peptide is LSGRSDNH (SEQ ID NO:5), LSGX (SEQ ID NO:16) or LSGK (SEQ ID NO:16)

19. A prodrugged antibody according to claim 7, having a structure according to formula (IIa) to (IIh): ##STR00017## ##STR00018## ##STR00019##
Description



CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit under 35 U.S.C. .sctn. 119(e) of U.S Provisional Application Ser. No. 62/859,835, filed Jun. 11, 2019; the disclosure of which is incorporated herein by reference.

SEQUENCE LISTING

[0002] Incorporated herein by reference in its entirety is a Sequence Listing named "200428_SEQT_13223WOPCT_YC.txt," comprising SEQ ID NO:1 through SEQ ID NO:10, which include nucleic acid and/or amino acid sequences disclosed herein. The Sequence Listing has been submitted herewith in ASCII text format via EFS-Web, and thus constitutes both the paper and computer readable form thereof. The Sequence Listing was first created using PatentIn 3.5 on Jan. 1, 2019, and is approximately 16 KB in size.

BACKGROUND OF THE DISCLOSURE

[0003] This application relates to prodruggable antibodies, prodrugs thereof, and methods of making and using such antibodies and their prodrugs.

[0004] Therapeutic antibodies can be used to treat a variety of diseases, especially cancer and inflammatory conditions. Examples of therapeutic antibodies that have received marketing approval from regulatory authorities include ipilimumab (YERVOY.RTM.), nivolumab (OPDIVO.RTM.), trastuzumab (HERCEPTIN.RTM.), cetuximab (ERBITUX.RTM.), rituximab (RITUXAN.RTM.), infliximab (REMICADE.RTM.), and adalimumab (HUMIRA.RTM.). Generally, a therapeutic antibody--like other antibodies--acts by binding with high specificity for and affinity to its molecular target (the antigen), to initiate the cellular processes related to its therapeutic action.

[0005] A prodrug can be used to reduce a therapeutic agent's off-target side effects. A prodrug is a version of a therapeutic agent that is less active, but which can be converted at or near the target tissue or organ into the active therapeutic agent. Commonly, prodrugging is achieved by covalently attaching to the therapeutic agent a moiety that reduces its activity. Removal of the blocking moiety at the target site by a factor or agent found there--low pH, an enzyme, anoxia, etc.--restores the activity of the therapeutic agent. See, for example: Trouet et al. 2004, Stagliano et al. 2013, Rodeck et al. 2010, and Lauermann 2014.

[0006] As with the case of other therapeutic agents such as small molecule drugs, it is desirable that the side effects of a therapeutic antibody be prodrugged to reduce or eliminate its action on a tissue or organ other than the one targeted for disease treatment. Classically, an antibody is a Y-shaped dimeric protein, each dimer half consisting of two chains, a heavy and a light chain, as shown in FIG. 1. Commonly, the two dimer halves are identical and are covalently linked to each other by disulfide bonds. The binding interactions of an antibody with its antigen occur through the antibody's complementarity determining regions (CDRs), of which there are three (CDR1, CDR2, and CDR3) on each heavy and light chain, in the variable regions thereof, for a total of six. The heavy and light chain variable regions (labeled V.sub.H and V.sub.L, respectively in FIG. 1) are located near the amino terminus of each protein chain. Thus, the six CDRs are referred to as V.sub.H CDR1, V.sub.H CDR2, V.sub.H CDR3, V.sub.L CDR1, V.sub.L CDR2, and V.sub.L CDR3. Flanking the CDRs are four framework regions (FR1, FR2, etc.) that provide a scaffold for the CDRs, so that the heavy and light chain variable regions have the N-to-C arrangement FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0007] For prodrugging an antibody, the V.sub.H and V.sub.L regions are potential sites for attachment of the blocking moiety due to the presence there of the CDRs responsible for antigen interactions. For example, Polu and Lowman 2014 disclose prodrugging an antibody by attaching a masking peptide to the N-terminus of the light chain of an antibody. Other disclosures relating to the prodrugging of antibodies include: Stagliano et al. 2016, Williams et al. 2015, Lowman et al. 2014, Lowman et al. 2015b, and Daugherty et al. 2015. Specific antibodies that have been prodrugged include those against these antigens: EGFR (Desnoyers et al. 2013, Lowman et al. 2015a, Lowman et al. 2017), JAGGED 1/2 (West et al. 2015), interleukin-6 receptor (West et al. 2016a), tissue factor pathway inhibitor (Wang et al. 2016), CD3 (Dennis et al. 2016), PDL1 (West et al. 2016b), CD166 (West et al. 2016c), CD71 (Sagert et al. 2016b), PD1 (Tipton et al. 2017), and ITGA3 (Sagert et al. 2016a).

[0008] Krystek et al., U.S. application Ser. No. 16/103,654, filed Aug. 14, 2018, discloses prodrugged antibodies in which a framework amino acid has been replaced by a Cys that serves as an attachment site for a blocking moiety. The linker by which the blocking moiety is attached is cleavable at the site of intended action to release the un-prodrugged antibody.

[0009] Some of the documents discussed herein are cited by first author or inventor and year of publication. Their full bibliographic citations are listed in the REFERENCES section towards the end of this specification.

BRIEF SUMMARY OF THE DISCLOSURE

[0010] A cancer treatment that is the subject of intense current interest is immune oncology, in which a cancer patient's own immune system is mobilized to attack the cancer. Although the immune system's cytotoxic T cells can be activated to kill cancer cells, checkpoint inhibitors provide a negative feedback that prevents their activation. One checkpoint inhibitor is CTLA4, which is expressed by activated Tcells. The binding of CTLA4 to its ligand on an antigen-presenting cell initiates the negative feedback signal. The anti-CTLA4 antibody ipilimumab (YERVOY.RTM.) has been shown to turn off the inhibitory mechanism by binding to CTLA4, thereby allowing cytotoxic T cell activation and killing of cancer cells. Ipilimumab has been approved for treatment of cancers such as melanoma. As with other therapeutic antibodies, it is desirable that the activity of an anti-CTLA4 antibody be confined to the site of intended action. One method for such confinement would be prodrugging.

[0011] The highly variable amino acid sequences in the CDRs accounts for the ability of antibodies to bind to huge variety of antigens, ranging from peptides to carbohydrates to small molecules of non-biologic origin to even metals. Because of the highly specific nature of the binding interactions of their amino acids with the antigen, the CDR amino acids of an antibody are disfavored candidates for replacement by another amino acid for prodrugging purposes, lest the binding interactions be disrupted. Thus, prodrugging efforts have focused on modifications at the amino terminus or in a framework region, as discussed above.

[0012] We have discovered that, unexpectedly, replacement of certain CDR amino acids in an anti-CTLA4 antibody with a Cys does not obviate the antibody's ability to bind CTLA4, but yet provides a chemical functionality for attachment of a cleavable blocking moiety to create a prodrugged antibody.

[0013] Thus, in one embodiment, there is provided an anti-CTLA4 antibody having [0014] (i) a heavy chain CDR1 comprising the sequence of amino acids of SEQ ID NO:1, [0015] (ii) a heavy chain CDR2 comprising the sequence of amino acids of SEQ ID NO:2, [0016] (iii) a heavy chain CDR3 comprising the sequence of amino acids of SEQ ID NO:3, [0017] (iv) a light chain CDR1 comprising the sequence of amino acids of SEQ ID NO:4, [0018] (v) a light chain CDR2 comprising the sequence of amino acids of SEQ ID NO:5, and [0019] (iv) a light chain CDR3 comprising the sequence of amino acids of SEQ ID NO:6; wherein one of Xaa at position 8 of SEQ ID NO:2, at position 5 of SEQ ID NO:4, at position 8 of SEQ ID NO:4, at position 3 of SEQ ID NO:5, or at position 7 of SEQ ID NO:5 is Cys. (The remaining Xaa's being other than Cys.)

[0020] In a first embodiment of the aforesaid antibody, Xaa at position 8 of SEQ ID NO:2 is Cys. In a second embodiment of the aforesaid antibody, Xaa at position 5 of SEQ ID NO:4 is Cys. In a third embodiment of the aforesaid antibody, Xaa at position 8 of SEQ ID NO:4 is Cys. In a fourth embodiment of the aforesaid antibody, Xaa at position 3 of SEQ ID NO:5 is Cys. In a fifth embodiment of the aforesaid antibody, Xaa at position 7 of SEQ ID NO:5 is Cys.

[0021] In one embodiment, there is provided a prodrugged antibody according to formula (I)

(BM-L).sub.m-Ab (I)

wherein

[0022] Ab is an antibody having [0023] (i) a heavy chain CDR1 comprising the sequence of amino acids of SEQ ID NO:1, [0024] (ii) a heavy chain CDR2 comprising the sequence of amino acids of SEQ ID NO:2, [0025] (iii) a heavy chain CDR3 comprising the sequence of amino acids of SEQ ID NO:3, [0026] (iv) a light chain CDR1 comprising the sequence of amino acids of SEQ ID NO:4, [0027] (v) a light chain CDR2 comprising the sequence of amino acids of SEQ ID NO:5, and [0028] (iv) a light chain CDR3 comprising the sequence of amino acids of SEQ ID NO:6; wherein one of Xaa at position 8 of SEQ ID NO:2, at position 5 of SEQ ID NO:4, at position 8 of SEQ ID NO:4, at position 3 of SEQ ID NO:5, or S at position 7 of SEQ ID NO:5 is Cys;

[0029] BM is a blocking moiety that inhibits binding of Ab to its antigen;

[0030] each L is, independently, a linker moiety bonded to BM and Ab, L comprising a cleavable moiety and being bonded to Ab at a Cys at position 8 of SEQ ID NO:2, at position 5 of SEQ ID NO:4, at position 8 of SEQ ID NO:4, at position 3 of SEQ ID NO:5, or at position 7 of SEQ ID NO:5; and

[0031] m is 1 or 2.


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