See also purification of bi-specific antibodies under particular antibodies purified

In general, IgG type bispecific antibodies are composed of two types of H chains (an H chain for antigen A and an H chain for antigen B and two types of L chains a L chain for antigen A and a L chain for antigen B). When such IgG type bispecific antibodies are expressed, 10 types of combinations are possible as combination of H2L2 since two types of H chains and two types of L chains are expressed. Among these, there is one type of combination that has the desired binding specificity for antigeb A on one arm and antigen B on the other arm. Consequently, in order to aquire the desired bispecific antibody, it is necessary to purify one type of antibody of interest from among ten types of antibodies, which is extremely low in efficiency and difficult. (Kuramochi (US 2014/0370020)

Since in bispecific antibodies the two H chains as well as the 2 L chains are different and can randomly associate, expression of these four chains leads to the formation of 10 different antibody variants. Correct H chain association resulting in a heterodimeric Fc can be enforced wuing KiH technology by introducing a builky tryptophan (Trp) resiude in one Fc fragment and forming a corresponding cavity on the other Fc fragment that can accommodate the Trip residues. More recently, multip alternative approaches to enable correct H chain association have been described such as relying on charge interactions. Although kiH technology was developed in the late 1990s, enabling correct L chain assocaition remained a major problem, and the only approach to achieve this at the time relied on the sue of common L chains for both specificities. However, the use of a common light chain required the de novo identificaiton of the corresponding antibody pairs, which can be challenging and/or time consuming depending on the desired target, and restrict the availabity and diversity of antibodies that can be sued; those methods allowing the generation fo bispecific antibodies from pre-existing antibody pairs were highly desired. (Keiine “Ten years in the making: applicaiton of CrossMab technology for the development of therapeutic bispecific antibodies and antibody fusion proteins” 2021, MABS, 13(1)).

Common Heavy chain with two light chains:

lamda/kappa bodies (common heavy change, one Kappa and one lambda): 

Fischer (US2014/0179547) discloses the generation of bispecific antibodies where two antibodies having different specificties sharing the same variable heavy chain domain but different variable light chain domains are isolated. The variable heavy chain domain is fused to the constant region of a heavy chain, one light chain variable domain is fused to a Kappa constant domain and the other variable light chain domain is fused to a Lambda constant domain. Preferably, the light chain variable domain fused to the Kappa constant domain is of the Kappa type and the light chain variable domain fused to the Lambda constant domain is of the Lambda type. However, it is also possible to generate hybrid light chains so that two variable light chain domains of the same type can be used to generate bispecific antibodies. The three chains are co-expressed in mamalian cells leading to the assembly and secretion in the supernatant of a mixture of 3 antibodies; two monospecific antibodies and one bispecific antibody carrying two different light chains. The antibody mixture is purified using standard chromatography techniques used for antibody purification. See antibody purification

Introduction:

A problem with KiH technology remains correct light chain associrion. While a common light chain can be used, a common light chain requires the de novo identification of the corresponding antibody pairs, which can be challenging and/or time-consuming depending on the desired target and restricts the avaiability and diversity of antibodies that can be used. CrossMab technology enables this correct light-chain association in bispecific antibodies. By incorporating the original heavy chain VH-CH1 domain in the Fab of the second specificity of the bispecific antibody as the novel “lgiht chain” and the original light chain VL-CL domain for the novel “heavy chain” by fusing them to the hing region of the Fc fragment, correct lgiht-chain association can be enfored. The Crossmab technology has evolved in the past decade into one of the most mature, versatile and broadly applied technologies in the field for teh generation of various bispecific antibody formats. (Kleine “Ten years in the making: applicaiton of CrossMab technology for the development of therapeutic bispecific antibodies and antibody fusion proteins” 2021, MABS, 13(1)). 

Roche developed the CrossMab approach as a possibility to enforce correct light chain pairing in bispecific hterodimeric IgG antibodies, when combining it with the KH technology. In this format, one arm of the intended bispecific antibody is left untouched. In the second arm, the whole Fab region, or the VH-VL domains or the CH1-CL domains are exchanged by domain crossover between the H and L chain. As a consequence, the newly formed “crossed” L chain does not associated with the (normal, i.e., not-crossed) H chain Fab reigon of the other arm of the bipsecific antibody any longer. Thus, the correct L chain aossication can be enfored by this minimal change in domain arrangement. (Schlothauer, WO 2016/071377)

Two problems must be solved to produce the desired bispecific antibody exclusively: effective induction of heterodimerization of the two heavy chains and discrimination between the two light- chain/heavy-chain interactions. The former can be overcome by introducing large amino acid side chains into the CH3 domain of one heavy chain that fit into an appropriately designed cavity in the CH3 domain of the other heavy chain [the “knobs into holes” (KiH) The latter problem is more difficult to address, because a total of four possible pairings of heavy and light chains remain, with only one of which represents the desired compound. To solve this, the heavy chain 1 and light chain 1 are unmodified. On the opposite side, a new “heavy” chain 2 consisting of an Fc part and the Fab of the original light chain. As the new “light” chain 2 the heavy chain domains VH and CH1 are used. Because the sequence of the modified heavy chain now is crossing over between light- and heavy-chain domains, the term “crossover” for this kind of domain interchange and the term “CrossMab” for antibodies based on this technology. Hetero- dimerization of the two heavy chains is achieved by using the KiH method. As a consequence of this domain rearrangement, associations between unrelated partners can no longer occur. The new “light” chain 2 on the crossover side consists of heavy-chain domains only; thus it cannot assemble with the remaining original heavy chain 1. On the other hand, the original light chain 1 on the unmodified side cannot interact with the new “heavy” chain 2 on the crossover side, because both partners contain the same light- chain heterodimerization interfaces, which do not interact. A bispecific antibody (Fig. 1C) with correct light-/ heavy-chain pairing in both Fabs and almost no deviation from the original IgG is thus obtained. (Klein, “Immunoglobulin domain crossover as a generic approach for the prouciton of bispecific IgG antibodies”. PNAS, July 5, 2011, 108(27). 

Applications:

–Autoimmune diseases:

Bispecific CrommMab based antibodies have been generated with the goal of treating autoimmune diseases. Fischer showed that combined inhibition of TNFalpha and IL-17 was more effective in inhibting the development of inlammation and bone and carilage destruction in arthritic mice compared to the respective monotherapies. For this purpose bispecific TNFalpha/IL-17 1+1 and 2+2 CroossMab (HC1-CL) antibodies were rpepared that showed superior efficacy in blocking cytokine ahd chemokine resosnes in vitro. (Kleine “Ten years in the making: applicaiton of CrossMab technology for the development of therapeutic bispecific antibodies and antibody fusion proteins” 2021, MABS, 13(1)).

–Cancer therapy and opthalmology:

For many years, anti-angiogenesis approaches blocking the vascular endothelial growth factor-A (FEGF-A) have been a major area of targeted cancer therapy. One of the first IgG based antibodies and the first bispecific CrossMab to enter clinical trails, in 2012, was the hterodimeric 1+1 VEGF/Ang-2 CrossMab (HC1-CL) vanuucizumab (RG7221) tarteting the pro-angioneic ligands VEGF-A and angiopoietin-2 (Ang-2), which are invovled in tumor angioenesis. VEGF and Ang-2 ahve also been shown to play an important role in ocular angionesis in diseases liek wet age related macular degerenation a. (Kleine “Ten years in the making: applicaiton of CrossMab technology for the development of therapeutic bispecific antibodies and antibody fusion proteins” 2021, MABS, 13(1)).

Another major field in targeted cancer therpay has been and continues to be apoptosis induction through death receptor (DR) signlaing. As conventional DR5 anitobdies have not been successful in clinical trails, approaches for tumor targeted DR5 agonism have been persued. Expression of the fibroblast activaiton prtoein (FAP) on tumor fibroblasts is found in the majority of solid tumors, making fAAP an attractive antigen for tumor targeting. Based on this rationale, FAP targeted bispecific antibodies and fusion proteins have been created using CrossMab technology that rely on FAP binding with one moiety to induce, with their second moeity, hyper-clustering of TNF receptor superfamily members like DR5 for apoptosis induction. (Kleine “Ten years in the making: applicaiton of CrossMab technology for the development of therapeutic bispecific antibodies and antibody fusion proteins” 2021, MABS, 13(1)).

With the advent of cancer immunotherpay and checkpoint inhibitor antibodies during the past decase, the development of bispecific antibodies for immunotherapy has attracted substantial attention in industry and academia, wehreas the interest in anti-angioenic and pro-apoptotic therapies has declined. In this context, bispecific monovalent dual checkpoint inhibitory PD-1 antibodies co-targeting the checkpoint inhibitory receptors TIM-2 or LAG-3 ahve been designed based on a bispecific 1+1 CrossMab (VH-VL+/-) format, allowing avidity mediated selectivity gain and thus enhanced selectivity for PD-1+ nad PD-1+TIM=3+/LAG-3+ double positive T cells. (Kleine “Ten years in the making: applicaiton of CrossMab technology for the development of therapeutic bispecific antibodies and antibody fusion proteins” 2021, MABS, 13(1)).

Many of bispecific antibodies currently being developed are bispecific T-cell engarers. One of the first IgG based and Roche’s firt, T cell bispecific antibody (TCB) to enter clinical trails was  the heterodimeric and invalent CEA/CD3E 2+1 TCB bisatamab. It is a hterodimeric CEA/CD3e bispecific antibodyin the 1+1 CrossMab (CH1-C2) format to whcih a single additional Fab targeting CEZ is fused to the N-terminus of the nokb containg H chain. (Kleine “Ten years in the making: applicaition of CrossMab technology for the development of therapeutic bispecific antibodies and antibody fusion proteins” 2021, MABS, 13(1)).

The msot advanted 2+1 T cell bispecific antibody is glofitamab which, in constrat to cibisatamab, is based on a 2+1 CrossMab (VH-VL) formal with charge interactions using vriable regions dervied from obinutuzumab. (Kleine “Ten years in the making: applicaiton of CrossMab technology for the development of therapeutic bispecific antibodies and antibody fusion proteins” 2021, MABS, 13(1)).

Central nervous system diseases:

Th treatment of CNS diseases with mAbs is hampered by the low penetration of antibodies thorugh the blood brain barrier. To overcome this limitation, Nieoether generated transferrin receptor targeted bispecific antibodies that allowed delivery of these antibodies through the blood brain barrrier and showed improved brain exposure and prevented plaque formation. Using this approach, BS-GANT was generated based on the amyloid-beta antibody gantenerumab as a trivalent C terminally fused amyloid-beta-TfR 2+ 1 bispecific antibodies in a 2+1 CrossMaby (VH-VL=/-) format with charges. (Kleine “Ten years in the making: applicaiton of CrossMab technology for the development of therapeutic bispecific antibodies and antibody fusion proteins” 2021, MABS, 13(1)).

–Viral Infections:

The appliction of CrossMab technology ahs become popular for the geenraiton of bispecific and multispecific antibodies targeting various viruses. Curing the past years, multiple highly potent bispecific antibodies targeting HIV-1 have been generated using CrossMab technoloyg for the prevention and treamtent of HIV-1. (Kleine “Ten years in the making: applicaiton of CrossMab technology for the development of therapeutic bispecific antibodies and antibody fusion proteins” 2021, MABS, 13(1)).

Introduction of difference in the charges to Heavy chains:

If two heavy chains and two light chains are expressed, there are 10 possible heavy chain and 10 possible light chain combinations. Accordingly, introducing difference in the charges of two heavy chains has been used to facilitation purification of BiAb. (“Proprietary Innovative Antibody Engineering Technologies in Chugai Pharmaceutical” 12/18/2012).

Knobs-into-holes strategy:

The homodimerization of the two heavy chains in an IgG is mediated by the interaction between the CH3 domains alone. Heavy chains were first engineered for heterodimerization in the 1990s using a “knowbs-into-holes” strategy. Starting fromm the a “knob” mutation (T366W) that disfavors CH3 homodimerization, compensation “hole” mutations (T366S, L368A, and Y407V) were identified by phage display providing efficient pairing witht the “knob” while disfavoring homodimerization. (Spies, Molecular Immunology 67 (2015) 95-106). 

Bascially, the concept relies on modifications of the interface between the two CH3 domains of the two H cahins of an antibody where most interactions occur. A bulky residue is introduced inot the CH# domain of one antibody H chain and acts similarly to a key (“knob”). In the other H chain, a “hole” is formed that is able to accommodate this builky residue, mimicking a lock. The resulting heterodimeric Fc region can be further stabilized by the introduction of artificial disulfide bridges. Notably, all KiH mutations are buried within the CH3 domains and not “visible” to the immune system. Correct H chain assocaition with hterodimierizaiton yield above 97% can be acheived by introducing xix mutaitons: S354C, T366A in the “knob” H chain and Y349C, T366S, L368A and Y407V in the “hole” H chain. (Schlothauer, WO 2016/071377). 

In the knobs in holes technology, one or more small amino acid side chains from the interfact of the first antibody molcule are replaced with larger side chains (e.g., tyrosine or tryptophan) (knobs or protuberances). The proteuberance may exist in the original interface or may be introduced synthetically. Compensatory “cavities” (holes) of identical or similar size to the large side chains are created on the on the interact of the second antibody molecule by replacing large amino acid side chainswith smaller ones. (e.g., alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-proudcts such as homodimers. (Giese, US 16/221,369 published as US 2019/0256556)

Schlothauer (WO 2016/071377) disclsoes variant Fc regions that specifically bind to SpA and that do or do not bind to human FcRn. Thesse variant Fc region contain specific amino acid mutations in the CH2 domain whereas the CH3 domain is not changed with respect to protoein A binding. The mutations when used in the hole-chain of a heterodimeric Fc regioulfide brdige(s) and wherein the CH3 domain of the first and second polypeptide both bind or both do not bind to protein A and allow for the purification of the heterodimeric Fc region, i.e., the separation of the heterodimeric Fc region from the homodimeric Fc region by-produce (hole-chain-hold-chain dimer). Thus in one embodiment, the heterodimeric polypetide includes a first polypeptide which includes in N to C terminal direction at least a pottion of the hinge region, which includes one or more cysteine residues, a CH2 domain, a CH3 domain and a second polypetide that includes in the N to C terminal direction at least a protion of the hinge resgion which includes one or mroe cysteine residues, a CH1-domain and a CH3 domain, wherein the first polypeptide includes the mutations Y349C, T366S, L368A and Y407V (hole chain) and the second polypetide includes the mutations S354C and T366W (knob-chain) and wherein the first polypetpide (hole-chain) icludes the mtuations I253A or I253G and L314A or L314G or L314D, wherein the first and second polypetpidds are connected by dis

Introduction of + charged amino acids into H chain CH3 and – charged amino acids into L chain CH3: 

Zhang (WO 2017/034770) discloses modifying antibody CH3 domain to promote heterodimerization between a first CH3 polypeptide and a second CH3 harboring polypeptide. In particular, at least two positively charged amino acids such as arginine, lysine or histidine are introduced into the first polypeptide by amino acid substitution to increase the positive charge while at least 2 negatively charged amino acids such as aspartic acid or glutamic acid are introduced into the seocnd polypeptide to increase negative charge of the polypeptide chain. Those different modifications at the itnerfact of two complementary CH3 omains presumably provides much stronger electrostatic attractions between those two chain to favor heterodimer formation rather than homodimer formation. In addition, cysteine molecules may be intoduced togehter wtih the charged amino acids to the appropriate position of both interfaces of the two complementary CH3 domains to allow inter-chain disulfide bond formation and further strenghten the heterodimer formation. In cerain embodiments, the molecule may be a bispecific, trispecific or qudrospecific antibody. 

Modification of H chain CH1 and L chain constant region;

Kuramochi (US 2014/0370020) discloses regulation of the association of the H and L chain such that in one eobmdiment the amino acids repel electrically or in a separate embodiment the amino acids do not repeal electrially. By making amino acid residues at given locations in the H constant region (CH1) and L chain constant region of a desired bomination that do not mutually repel electrically, a desired combination of H and L chain can be foremd by suing the attractive force of the electric charges. Amino acids that do not mutually repeal electrially include amino acids in which one of the amino acids is a positively charged amino acid and the other is a negatively charged amino acid. 

Modifying AAs of first and/or second polypeptides to create differences in isoelectric point:

Igawa (US 2009/0263392) discloses modifing both or either one of a nucleic acid encoding the amino acid residue of the first polypetpide and a nucleic acid encoding the amino acid residues of the second polypeptide, usch that the difference between the isoelectirc point of the rift and second polypeptide will be incdreased. The modification results in peaks of the homomultimer for the first polypeptide and second polypeptide and heteromultimer of the first polypeptide and the second polypeptides that can be seaprated. 

Common Light chain with two Heavy Chains:

If two heavy chains and two light chains are epxressed, there are 10 possible H and 10 possible L chain combination. But if a common light chain is used for two H chains, combinations become only three. (“Proprietary Innovative Antibody Engineering Technologies in Chugai Pharmaceutical” 12/18/2012)

A common light chain strategy was applied to assemble IgG like bispecific antibodies which can be combined with the knobs-into-holes approach. The mechanism of a common light chain is based on the fact that antibodies discvoered form phase display screening against diverse antigens often share the same VL domain, reflecting the very limited size of the L chain repertoire in the phage library. One of the advantages of the common L chain format is that it allows the use of methods that simplify the antibody engineering and the purificaiton process in industrial production. (Wang, “Design and Production of Bispecific Antibodies” Antibodies, 2019)