Method of manufacturing bispecific antibodies, bispecific antibodies and therapeutic use of such antibodies

A technology of bispecific antibodies and immunoglobulins, applied in chemical instruments and methods, antibody mimics/scaffolds, anti-animal/human immunoglobulins, etc., can solve the problem of increasing the risk of inducing anti-drug antibodies and failing to mass-produce Issues such as clinical development and commercialization, increasing costs

Pending Publication Date: 2020-01-24
NOVARTIS AG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

This in vitro shuffling is a complex additional procedural step that requires careful validation and analytical evaluation and can significantly increase costs
[0009] Thus, existing methods for generating bbmAbs may limit the diversity of antibody repertoires available for screening binders, or may not provide sufficient overall yield, purity, and product quality in a sufficiently cost-effective manner to allow large-scale Manufactured for clinical development and commercialization
Additionally, any modification of the protein chain inherently increases the risk of inducing anti-drug antibodies

Method used

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  • Method of manufacturing bispecific antibodies, bispecific antibodies and therapeutic use of such antibodies
  • Method of manufacturing bispecific antibodies, bispecific antibodies and therapeutic use of such antibodies
  • Method of manufacturing bispecific antibodies, bispecific antibodies and therapeutic use of such antibodies

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0192] 5. Example 1: Production of bbmAb bbmAb1

[0193] By way of example, the following describes the generation of specific bbmAbs to enable those skilled in the art to practice the invention.

[0194] The resulting bbmAb, bbmAb1, is a bispecific IgG1 with a LALA silent mutation that simultaneously binds to two distinct targets, IL-1β and IL-18. The antibodies bind two distinct antigen-binding arms (Fab fragments), while the Fab against IL-1β is based on mAb2 and contains a kappa light chain (Vk6). The Fab against IL-18 is based on mAb1 and consists of a lambda light chain (Vλ1). To drive heterodimerization of the Fc domain during expression, "knobs" with large amino acid (aa) side chains (S354C and T366W) and small aa side chains (Y349C, T366S, The "holes" of L368A, Y407V) were introduced into the mAb2 heavy chain.

[0195] For ease of reference, the CJM112 hypervariable region based on Kabat definition and Chothia definition, and V L and V H Domain and complete amino...

example 2

[0325] 6. Example 2: In vitro activity of bbmAb1

[0326] The binding activity of bbmAb1 was tested in various cellular assays.

[0327] (1) Materials and methods

[0328] (a) For solution equilibrium titration (SET) determination

[0329] The following materials were used:

[0330] Biotinylated recombinant human IL-18 (BTP25828)

[0331] Recombinant Cynomolgus IL-1β (Novartis)

[0332] SULFO-TAG labeled anti-human IgG antibody (Meso Scale discovery (Meso Scale discovery, MSD) #R32AJ-5)

[0333] Goat anti-human Fab specific antibody conjugated with MSD SULFO-TAG NHS ester (Jackson Immuno Research #109-005-097, MSD #R91AN-1), BSA (Sigma # A-9647)

[0334] MSD Read Buffer T with Surfactant (MSD#R92TC-1)

[0335] Phosphate Buffered Saline (PBS) 10x (Teknova #P0195) Tris Buffered Saline pH 7.5 (TBS) 10x (Teknova #T1680) Tween-20 (Fluka #93773)

[0336] Polypropylene microtiter plate (MTP) (Greiner #781280)

[0337] 384-well plate, standard (MSD#L21XA)

[0338] (b) For ce...

example 3

[0423] 7. Example 3: Effects of combined stimulation and blockade of IL-1β and IL-18 in PBMCs

[0424] Inflammasome activation-dependent cleavage of effector cytokines IL-1β and IL-18 results in the induction of secondary pro-inflammatory mediators that promote immune cell recruitment / activation not only systemically but also at sites of inflammation. In two different mouse models of lethal systemic inflammation (a) LPS injection model and (b) FCAS mice (activating missense mutations in NLRP3), with single IL-1β or single IL-18 absent / inhibited In contrast, simultaneous absence / inhibition of IL-1β and IL-18 was more protective against lethality, demonstrating an additive or synergistic mechanism of immune activation (Brydges 2013, van den Berghe 2014). bbmAb1 is a human / marmoset IL-1β / IL-18 reactive bispecific mAb with no rodent cross-reactivity and therefore could not be tested in mouse models. Therefore, we stimulated human PBMCs using LPS / IL-12 in vitro to mimic activation...

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Abstract

The invention relates to bivalent bispecific monoclonal antibodies (bbmAb) or variants thereof, and methods of manufacturing such antibodies by co-expressing modified Fc-5 mutated derivatives of two different monoclonal antibodies in mammalian cell lines.

Description

[0001] sequence listing [0002] This application contains a Sequence Listing filed electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy, created on May 30, 2018, is named PAT057716-WO-PCT_SL.txt and is 68,498 bytes in size. technical field [0003] The present invention relates to bivalent bispecific monoclonal antibodies (bbmAbs) or variants thereof, as well as said knob-into-hole modified, FC mutant derivatives to produce such antibodies. Background technique [0004] Bispecific antibodies, ie antibodies that bind two different epitopes, are well known in the art. One method of producing bispecific antibodies is the so-called knob-and-hole (KiH) method, such as described by Merchant et al., Nat. Biotechnol., 16:677-681 (1998), in which the Mutations such as Y349C, T366S, L368A, Y407V modify the first heavy chain IgG to display a socket-like structure; and by introducing point mutations S354C, T366W (Merchant et al., Na...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/00C07K16/08C07K16/46C07K16/24
CPCA61P1/00A61P9/10A61P9/14C07K16/00C07K16/244C07K16/245C07K16/2863C07K16/2887C07K16/46C07K16/468C07K2317/24C07K2317/31C07K2317/51C07K2317/515C07K2317/526C07K2317/53C07K2319/00
Inventor M·O·巴尔多夫T·布赫C·格拉夫D·海特曼T·约斯多克H-P·科诺普夫R·科勒J·科瓦里克S·J·奥利弗D·帕特尔M·沃伊塞特施雷格尔
Owner NOVARTIS AG
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