Antibody-SN-38 immunoconjugates with a CL2A linker

A CL2A-SN-38, -PABO-CO-20-O-SN-38 technology, applied in anti-animal/human immunoglobulin, anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, Antibodies, etc., can solve the problems of reducing the therapeutic window, non-existent therapeutic efficacy, discontinuity, etc.

Inactive Publication Date: 2016-03-16
IMMUNOMEDICS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

While the very stable binding to the MAb results in stability in circulation, the conjugate is also processed in the liver, spleen and kidney, releasing toxic drugs in these organs and potentially reducing therapeutic window
Although recent regulatory approval (brentuximabvedotin) for Hodgkin lymphoma and approval (ado-trastuzumab emtansine) is encouraging for refractory breast cancer, but maximum administrable doses of calicheamicin conjugates in non-Hodgkin's lymphoma The absence of therapeutic efficacy and its subsequent discontinuity, and the market withdrawal of gemtuzumab ozogamicin for AML point to limitations in the use of ultratoxics in ADCs

Method used

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  • Antibody-SN-38 immunoconjugates with a CL2A linker
  • Antibody-SN-38 immunoconjugates with a CL2A linker
  • Antibody-SN-38 immunoconjugates with a CL2A linker

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0402] Example 1: Preparation of CL2A-SN-38

[0403] A preferred reaction scheme for the synthesis of CL2A-SN-38 is shown in figure 1 , including the following steps for an improved method for large-scale production.

[0404] O-(2-azidoethyl)-O’-[(N-diglycolyl-2-aminoethyl base)-Lys(MMT)-PABOH]heptaethylene glycol (intermediate 3, figure 1 ) preparation: In a 500-mL single-necked flask, add commercially available Fmoc-Lys(MMT)-OH (16 g), p-aminobenzyl alcohol (3.26 g), and EEDQ (6.52 g), followed by anhydrous dichloromethane (80mL). After stirring overnight, diethylamine (25 mL) was added and after a further 6 hours the reaction mixture was concentrated to a volume of ~50 mL. It was diluted with heptane, and the solution was concentrated back to 50 mL. Two additional chases with heptane (50 mL each) provided a biphasic mixture with a gummy material at the bottom. The gummy material was dissolved in dichloromethane (24 mL), stirred, and worked up to slowly add heptane ...

Embodiment 2

[0408] Example 2. Conjugation of CL2A-SN-38 to antibodies

[0409] Anti-CEACAM5 humanized MAb, hMN-14, anti-CD22 humanized MAb, hLL2, anti-CD20 humanized MAb, hA20, anti-EGP-1 humanized MAb, hRS7 and anti-mucin humanized MAb The derivatized MAb, hPAM4, was used in these studies. Gently reduce each antibody with tris(2-carboxyethyl)phosphine (TCEP) in phosphate buffered saline at a pH in the range of 7-7.4, adjust the pH to 6.5, and use 5-10% v / v was reacted with DMSO as a co-solvent with ~10-fold molar excess of CL2A-SN-38 and incubated for 20 min at ambient temperature. A 10-fold molar excess of N-ethylmaleimide relative to the antibody was used as an aqueous solution to block any excess thiols.

[0410] The conjugate was purified by tangential flow filtration (TFF) using 20-30 diafiltration volumes of final formulation buffer 25 mMMES, pH 6.5. This method avoids tedious serial purifications on size-exclusion and hydrophobic columns, thus enabling the purification of hund...

Embodiment 3

[0413] Example 3. In vivo therapeutic efficacy in preclinical models of human pancreatic or colon cancer

[0414]Immunocompromised athymic female nude mice bearing subcutaneous human pancreatic or colon tumor xenografts were treated or left untreated with specific CL2A-SN-38 conjugates or control conjugates. The therapeutic efficacy of the specific conjugates was observed. figure 2 shows the Capan1 pancreatic tumor model in which specific CL2A-SN-38 conjugates of the hRS7 (anti-EGP-1), hPAM4 (anti-mucin) and hMN-14 (anti-CEACAM5) antibodies showed higher - Better efficacy of SN-38 conjugate (anti-CD20) and untreated control. Similarly, in the BXPC3 model of human pancreatic cancer, specific hRS7-CL2A-SN-38 showed better therapeutic efficacy than control treatment ( image 3 ). Likewise, treatment with specific hMN-14-CL2A-SN-38 was more effective than non-treatment in an aggressive LS174T model of human colon cancer ( Figure 4 ).

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Abstract

The present invention concerns improved methods and compositions for preparing SN-38 conjugates of proteins or peptides, preferably immunoconjugates of antibodies or antigen-binding antibody fragments. More preferably, the SN-38 is attached to the antibody or antibody fragment using a CL2A linker, with 1-12, more preferably 6 or less, most preferably 1-5 SN-38 moieties per antibody or antibody fragment. Most preferably, the immunoconjugate is prepared in large scale batches, with various modifications to the reaction scheme to optimize yield and recovery in large scale. Other embodiments concern optimized dosages and / or schedules of administration of immunoconjugate to maximize efficacy for disease treatment and minimize side effects of administration.

Description

[0001] related application [0002] This application claims priority to US Patent Application Serial No. 13 / 948,732, filed July 23, 2013. [0003] sequence listing [0004] This application contains a Sequence Listing that has been filed electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on April 4, 2014, is called IMM340WO2_SL.txt and is 60,597 bytes in size. field of invention [0005] The present invention relates to therapeutic immunoconjugates having an improved ability to target various cancer cells, infectious disease organisms and / or for the treatment of autoimmune diseases, said conjugates comprising an antibody moiety and a drug selected from The drug moiety of the camptothecin group. The antibody is linked to the drug moiety via an intracellular cleavable linkage that enhances therapeutic efficacy. Most preferably, the camptothecin is SN-38 and the linker linking the antibody to the drug moiety is CL2...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K31/4745A61K39/395A61K47/48
CPCA61K38/395A61K39/39558A61K45/06A61K31/337A61K31/4184A61K31/4375A61K31/454A61K31/513A61K31/675A61K31/7088A61K31/713C07K16/2803C07K16/2833C07K16/2887C07K16/30C07K16/3007A61K2039/505A61K2039/507A61N2005/1098C07K2317/77C07K2317/92C07K2317/94A61K47/6803A61K47/6851A61K31/4745A61K2300/00
Inventor S.V.戈文丹J.B.盖尔N.J.霍尔曼D.M.戈登伯格
Owner IMMUNOMEDICS INC
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