See “homeostasis and half life” of antibodies  See also FcyR structure See also fc receptors under signal transduction

web database of mAbs with clinical indications 

supplies: Quickchange mutagenesis kit (Agilent Technologies)

software: Cn3D4.3.1 (can view 3D structures from NCBIs Entrez Structure database.

The pharmacokinetic requirements of a given antibody depend upon the nature of its application. In the case of therapeutic antibodies, which generally act either by inhibiting a signlaing pathway or by inducing antibody-dependent cell mediated cytotoxicity (ADCC) or complement dependent cytotoxicity (CDC), a prolonged serum half-life is desirable, for longer bioavailability and to avoid repeated injections. Alternatively, if an antibody is being used as a vehicle for the delivery of a cytotoxic agent, the rapid elimination of the untargeted conjugate is preferably, to savoid non-target-itssue toxicity. In general, intact antibodies and antibody fragments greater than 60 kDa are above the renal threshold and exhibit polonged circulation, whereas small antibody fragments less than 60 k Da exhibit a shorter serum half life. (Jain et al. TRENDS in Biotechnology, 259&), 2007). 

The constant region of the heavy chain mediates the binding of the antibody i) to cells bearing a Fc gamma receptor (FcgammaR), such as phagocytic cells, or (ii) to cells bearing the neonatal Fc receptor (FcRn) as known as Brambell receptor and to some factors including factors of the classical complement system such as component (C1q). 

One of the key factors in choosing an IgG isotype for a therpaeutic antiboy or Fc fusion protein is how the therapeutic antibody or Fc fusion protein is how the antibody binds to Fcy receptors on immune cells and to complement factor C1q. The four human IgG isotpypes bind the activating Fcy recetpros, FcyRI, FcyRIIa, and FcyRIIIa, the inhibitory receptor FcyRIIb and C1q with different affinities, yielding very different activities. (William Strohl, Current opinion in Biotechnology, 2009, 20 585-691).

The binding of IgG to the activating (FcyRI, FcyRIIa, FcyRIIIa and FcyRIIIb) and inhibitory (FcyRIIb) FcyRs or the first component of complement (C1q) depends on residues located in the hinge region and the CH2 domain. Two regions of the CH2 domain are critical for FcyRs and coplement C1q binding, and have unique sequences in IgG2 and IgG4 (Eon-Duval, WO 2008/025747A1).

There are four known sublcasses of IgG molecules, IgG1, IgG2, IgG3 and IgG4. IgG4 molecules differ from the other IgG isotypes in that the disulfide bonds that link the two heavy chain subunits together do not always form (US004/0092719). Human IgG1 and IgG3 have strong ADCC effects while IgG2 exerts weak ADCC and CDC effects. Human IgG4 displays weak ADCC and no CDC effects( US2010/0190961). 

IgG2 residues: substitution of IgG2 residues at positions 233-236 into human IgG1 grealy reduced ADCC and CDC (Eon-Duval, WO 2008/025747A1). 

Altering Effector function (see Fc variants for Fc Receptor binding)

Antibody Variants with Increased or Reduced Half-Life (see also altering pH dependence of antibodies)

Altering FcRN-antibody affinity for improved pharmacokinetics

The neonatal Fc receptor (FcRn) has an impportant role in controlling the levels of serum IgGs by facilitating their recylcing and preventing their catabolishm in the lysosomes of the vascular endothelial cells. The pharmaacokinetics of IgG can be modulated by altering the binding affinities of the Fc region to the FcRn. Mutations resulting in an increased binding affinity of IgG to the FcRn translate into prolonged persistence whereas those that decrease the binding affinity reuslt in shorter half life of the IgGs in the serum. (Jain et al. TRENDS in Biotechnology, 259&), 2007).

Presta (US 5,739,277) discloses antibody variants such as Fab which includes a salvage receptor binding epitope of an Fc region of an IgG and thereby have increased circulatory half-life.

Engineering for increased stability -less aggregation:

Wang investigatied the aggregate-prone regions (APRs) of antibodies on the CDR region and found that the APRs most frequently appeared in CDR-H2 and less frequently in the CDR-H3. They also showed that aromatic residues (e.g., Tyr and Trp) are favored both on the CDR and APRs, indicating that co-incidence of APRs with CDR sites can potentially cause the loss of function upon aggregation. Trout also quantified the exposure of hydrophobic residues averaged over snapshots from MD simulations and they used this value as a novel indicator to predict APRs. They dientified 14 agregaiton prone motifs in const regions of human IgG molecules, which are not conserved among the other antibody classes. (Zhao, “In silico methods in antibody design” Antibodies, 2018)

Modulation of Effector Functions:

Besides antigen recognition, the effector functions, such as ADCC, CDC and antibody dependent cell mediated phagocytosis can also be engineered based on the structures between fragment crystallizable region (Fc) and receptors nad the computations techniques in a high throughput way. The engineerable parts are hte hinge region, CH2 domain, N-glycans and N-glycan attached residues. All’acqua introduced various modificaitons in the hinge region of mAb to modulate the hinge’s lenght, flexibility and or biochemical properties. They found that the upepr and middle hinge are important, and mutations introduced to these regions can modulate the FcyRIIIa or C1q binding. Lazar applied computational optimziation of the Fc region using Protein Design Automatation technology and sequence prediction algorithm. They created vairous mutations to improve the affintiy up to 169 fold with a FcyRIIIa:IIb ratio about ine-fole. Engineering ont he N-glycan can also modulate the effector fucntions. They glycans at Asn-297 (N-glycan) are important to maintain the quaternary structure and the stability of the Fc, as well as the Fc-Fc receptor recognition. The deglycosylation of IgG1 resulted in a 40 fold loss in FcyRI binding. (Zhao, “In silico methods in antibody design” Antibodies, 2018)

Altering Binding Affinity

Substitutions in the Human Framework regions

Foote (J Mol Biol. 1992, 224(2): 487-99) discloses that substitutions in the human framework regions are sometimes required for high affinity antigen binding and disclose an antigen binding by a reshaped antibody derived form the mouse anti-lysozyme antibody D1.3 and several variants in which point mutations were introduced in the framework positions to improve its affinity. 

Functional Enhancement through Fusion of Target-Specific Peptides

Zybody: A zybody is a full lengh mAb to which modular recognition domains (MRDs) are fused recombinatly via the N and C termini of the mAb heavy and light chains. MRDs are relatively small polypeptides sequences (typically less than 60 amino acids) selected for target specificity from combinatorial libraries. Such MRDs includes small, domain-based structures such as knottins or affibodies as well as unstructured (linerar) or disulfide-constrained peptides. Depending on the number of Muker RDs fused to the mAb scaffold, zybodies may be from bi to penta specific, which each of the specificity represented bivalently. Additional valencies can be achieved by fusion of a single MRD to multiple temini. The arming of a conventionall mAb with up to four additional specificities requires unique nomenclature that defines the mAb scaffold, MRD location and corresponding specificities. (Lafluer “Monocloanl antibody therpaeutics with up to five specificities: functional enhancement through fusion of target specific peptides” MABS 5(2), 208-218, 2013). 

Engineering of Structural Loops:

Strands of antiparallel beta sheets are connected by loops to form a compreseed antiparallel beta barrel. In the variable region, some of the loops of the domains contribute essentially to the specificity of the antibody (i.e., the binding to an antigen by the natural binding site of an antibody). These loops are called DCR loops. The CDR loops are located within the CDR loop region, which may in some cases include also teh variable framework region that is adjacent to the CDR loops. It is known that VFRs may contribute to the antigen binding pocket of an antibody, which generally is mianly detemreined by the CDR loops. Thus, those VFRs are considered as part of the CDR loop region. They would not be appropriate for engineering new antigen binding sites. Contrary to those VFs within the CDR loop region or located proximinal to the CDR loops, other VFRs or variable domains would be suitable for engineering. Those are the structural loops of the VFRs located opposite to the CDR loop region or at the C temrinal side of a vriable immunoglbouilin domain. (himmler (US 2010/0184615).

(HImmler (US 2010/0184615) disclsoes modification in the loops region sof CH1, CH2, CH3 and CH4. In one embodiment, they include at least one modificaiton within amino acids 7-22, 39-55, 77-89 or 89-104 according to IMGT. Modificaiton can result in a substitution, deletion and or insertion of one or more nucleotides or amino acids such as a point mutation or even exchagne of whole loops. 

Altering/modifying Isoelectric point of antibody:

Hattori (“Redcued elimination of IgG antibodies by engineering the variable region” Protein Engineering Design & Selection 23(5), 385-392, 2010) idscloses that a novel non-FcRn dependent approach to reduce the elimination of IgG antibodies. Pharmacokinetic studes conductted in normal mice of various humanized IgG4 antibodies, which had identical constant regions but different variable regions sequences, revelaed tha na antibody with a lower iselectric point (pI) has a longer half-life. These antibodies exhibited comparable binding affinity to FcRn and with the antibodies with lower pIs, a longer half-life was also observed in beta2 microglobulin knockout mice, suggesting that differences in the pharmacokinetics were due to a non-FcRn dependent mechanisms. Selected substitutions int he variable region without substitution int he Fc region lwoered the pI but did not reduce biologcial activity and showed a significant reduction in the clearance of the antibody in cynomolgus monkey. 

Igawa (US 2011/0076275) disclsoe smodifying the isoelectric point of an antibody while retaining its antigen binding activity by modifying the charge of at least one exposable amino acid resiude on the surface of the complementarity determining region (CDR). In some embodiments the antibody is a multispeific antibody wehre either one or both of the first a second polypeptides are modified. The antibody half-life in plasma is prolonged when its isoelectric point value is decreased. Conversely, the half-life is shortened when its pI is increased. Higher pI points are knonw to improve the transfer of antibodies into tissues. When a polypeptide of itnerest with controlled pharmacokinetics in plasma cannot be produced by slight modificaitons tot he surface charge, the desired antibody can be obtained by repeating modificaiton of surface charge and evaluation of pharmacokinetics in plasma. 

Igawa (Protein Engineering Design & Selection, 23(5), pp. 385-392, 2010) discloses  that an antibdoy with a lower isoelectric pint (pI) has a longer half-life. Based on these observations, they engineered the pharmacokinetic proeprties of a humanized anti-IL6 receptor (IgG1) antibody. Selected substitutions in the variable region without substitution in the Fc region, lowered the pI but did not reduce biologcial activity and showed a significant reduction in clearance of the antibody in cynomolgus monkey. 

Nespor (WO 2018/224950) discloses modifying the isoelectric point of an antibody by providing an antibody that includes a first polypeptide that incldues a H chain variable region and a second polypeptide that includes a H chain variable region and substituting in at least one of the frist and second polypeptides of the antibody one or mroe amino acid resiudes of the H chain VH region wehrein the substituting inccreases or decreases the isoelectrictric point of the antibody. 

Randomization of CH2 domain:

Ruker “Introducing antigen-binding sites in structural loops of immunoglobulin constant domains: Fc fragments with engineered HER2/neu-binding sites and antibody properties” Protein Engineering Design and Selection, 23(4), pp. 289-297) discloses that the CH2 domain of human IgG1 has been used as a library scaffold and isolated CH2 domains with specific binding affinity to a HIV-1 gp 120-CD4 complex has been described. The new binding site was formed by engineered sequences in teh BC and FG loops of the CH2 domain, located at the N-temrinal tip of the domain. However, isolated CH2 domains with mutations in these lops are unlikely to be able to elicit effector fucntions, since binding of the Fc fragment to Fc gamma receptors occurs in an asymmetric fashion in the lower hinge region and in the upper CH2 region and involves different residues from each of the chains of the Fc, including CH2 loop residues. 

Insertions/Randomization/Substitutions of CH3 region:

Guo (US Patent Application No: 17/877,612, published as US 20230041757) discloses that a polypetpide such as a single chain antigen-binding polypetpide such as a single cahin variable fragment (scFv) or a single-domain antibody (nanobdy) or even an entire functional protein such as a cytokine can be fused to to a particular region such as 344-382 of the CH3 domain. In some embodiments, Guo teaches a multispecific antibdoy that includes awhich includes a first antigen binding site that binds a first antigen and a second antigen binding site that specifically binds to a second antigen. In some embodiments, the second antigen binding site is located within a polypeptide such as a scFv or a nanobody fused to the multispecific antibody at the CH3 domain at a region from position 344-382 of the CH3 domain. This particular region (344-382 EU numbering) is referred to by Guo as the “3A site”. As compared to some other modified immunoglobulins, the immunoglobulins with this modification are very stable and the immunoglobulins with a non-native polypetpide fused at this site can be expressed at a high level and they do not form aggregates. This 3A site is located in the A and B strand, which seems to be important for the function and stability of the CH3 domain. Guo discloses all different comibnations of the starting amino acid and the ending amino acid at the 3A site. For example, if the non-native polypetpide is linked to the amino acid residues at position 343 and 383, the entire 3A site (positions 344-382) is replaced by the non-native polypeptide. If the non-native polypeptide is linked to two consecutive amino acid resiudes such as position 357 and 358, the non-native polypeptide is inserted between the two consecutive amino acid residues. The fusion of the non-native polypetpide at the 3A site does not interfere with the antigen-binding site of the immunoglobulins. In addition, the fused non-native polypetpide can maintain its bioactivity. Moreover, the modification does not significantly affect the binding affinity of Fc to FcyRIIA, FcyRIIIA, FcyRIIIB or FcRn receptors. Guo also teaches in some embodimetns the modificaitons can occur at other Fc sites such as the “3B”, “3C” and “3D” sites. For example, the 3D sites starts from position 413 and ends at position 422. Guo exemplfies construction of a fusion protein which incudes a mouse Il-7 domain fused to the 3A site of an anti-PD-L1 IgG1 antibody. The fusion site was located within the 3A site of the H chain CH3 domain of the PD-L1 antibody. Guo also discloses fusing a scFv haivng the VH and VL sequences of anti-PD-L1 antibody to the 3A site of an anti-PD-1 antibody. Guo also exemplifies fusing a PD-L1 antibody with the extracellular domain of human TGFbR2 at the H chain 3A site. Also disclosed are plasmids to express a fusion protein with anti-CD40 single chain variable fragment (scFv) fused at the heavy chain 3A site of a PD-L1 IgG1 antibody. 

Wilkinson discloses monomeric polypeptides such as a monomeric antibody having a H chain, a varaint Fc region and a light chain. The variant Fc regions has substitutions that inhibit dimer formation of the Fc region. In certain emboidmetns, the variant Fc region includes one or more amino acid substiuttions within or close to the CH3 interface of the Fc region such as amino acid positions 347-347, 349, 350, 351, 352, 354, 356, 357, 360, 362, 364, 366, 368, 370, 390, 392, 393, 394, 395, 396, 397, 398, 399, 400, 405-409, 411 and 439. The variant Fc region includes the one or more amino acid substitutions relative to the parent Fc region sequence that reduce or eliminate homodimerization between two Fc polypetpides, e.g., repelling substiutions. Examples of suitable repelling substiutions include that ahving a charged side chain, a large or builky side chain or a hydrophilic side chain. For example, an amino acid residue that does not have a positively charged side chain in the parent Fc sequence may be replaced with an amino acid haivng a positively charge side chain to form the variant Fc region.

AB loop (358-362) and EF loop (413-415, 418-419) of CH3 domain: (“Fcabs”)

Everett (“Generation of Fcabs targeting human and murine LAG-2 as building blocks for novel bispecific antibody therapeutics” Methods, 154, 2019, pp. 60-69) discloses Fcabs (Fc region wtih antigen binding) targeting human and murine LAG-3 which were generated from phage libraries. The Fcabs bind to LAG-3, ionhibiting its interaction with MHC class II and induce IL-2 production in a T cell assay. Lymphocyte-activation gene (LAG-3) is a member of the immunoglobulin superfamily that is expressed on activated T cells, NK cells, gameepisolon T cells, activated B cells and plasmacytoid DCs. It has been reported to induce immune syuppression, in part through persistent high expression on a proportion of regulatory T cells. LAG-3 is related to CD4. The mAb is a bispecific antibody in which the Fc region of a mAb has been replaced with an Fc region with antigen binidng (Fcab). Fcabs contain engineered loops in their CH3 domains that introduce a novel antigen binding site. Randomisation of residues in the AB and EF loops (residues 11-18 and 92-101 by IMGT numbering) generates a continuous binding surface at the distal end of the CH3 domain without adding extra amino acids at the antibody C temrinus, thusmaintaining the integrity of the Fc fragment structure. The incorporation of an Fcab into a conventional mAb generates a mAb2, a mAb-like molecul with two additional antigen binding sites. 

Ruker/Wozniak-Knopp ( “Introducing antigen-binding sites in structural loops of immunoglobulin constant domains: Fc fragments with engineered HER2/neu-binding sites and antibody properties” Protein Engineering Design and Selection, 23(4), pp. 289-297) discloses yeast surface display libaries of human IgG1 Fc regions in which loop sequences at the C-terminal tip of the CH3 domain were randomized. Sorting these libraries by FACS for binders against HER2/neu yielded antigen-specific Fc binders of which one was affinity matured, resulting in a clone which showed greater than 10 fold improvement in antigen-binding activity versus the parental clone. The regions chosesn by Ruker for randomization are located in teh EF and in the AB loops. Both of these loops contain short alpha-helical stretches. Their replacement by random sequence allowed establishment of libraries form which stable folding and well expressing Fc mutants with engineered binding sites could be selected with high efficiency. Ruker’s choice was positions 358-362 in teh AB loop and 413-415 as well as 418-419 in the EF loop. The choice for randomizaiton was based on the criteria that side chains should be well exposed to the solvent and they should be as structurally indepndent as possible form teh body of the Fc fragment and the individual surfaces of the two loops together should form a choherent surface patch on the tip of the CH 3 domain, creaitng the complete engineered binding site. The yeast S. cresisia was sued for displaying the library due to the fact that the Fc fragment is a glycoprotein which naturally fomrs a disulfidelinked homodimer. Ruker created a large yeast display library of engineered Fc fragments, containing randomized positions and insertions in the AB and in the EF loops and isolated Fc fragments, which bind specifically and with high affinity to HER2/neu. Such Fc fragments were termed “Fcab“, i.e., Fc antigen binding (Fcab is a trademark of f-star Biotechnologische Forschugns). Ruker discloses that the Fc fragment of IgG1 is an ideal alternative small size antibody format. With the exception of the antigen-bnding site, it has all the attractive properties of a complete antibody, i.e., the ability to elicity effector functions via binding to Fc gamma receptors and to the complement activator C1q, as well as the long half-life of antibodies mediated through binding to FcRn. The loop regions, which are ideally suited for the generation of antigen-binding sites, are the ones located at the C terminal tip of the CH3 domain and include loops AB, CD and EF.  Ruker chose positions 358-362 in the AB loop, 413-415 as well as 418-419 in the EF loop for randomization. 

Lobner, (“Engineered IgG1-Fc–one fragment to bind them all” Immunological Reviews, 2016) discloses that the Fc of an existing mAb can be replaced with an Fcab to generate a so-called mAb2 that bivalently binds to one antigen via its Fab fragments and mono- or bivalently to a second antigen via the Fcab porition. 

(EP2546268, published 1/16/2013) discloses modula antibodies based on an Fc molecule having binding strcutures in the structural loop region, such as a non-CDR binding site in the C terminal structural loop region of one or both CH3 domains, a so call “Fcab” molecule (Fc antigen binding). The modular antibody include an immunoglobulin domain with a specificity to bind at least a receptor on a tumor cell and an immunoglobulin domain with a specificity to being at least an STAG, to obtain an immunoglobulin with at least both sepcificites. The term “soluble tumor associated antigen” or “sTAG” refers to antigens or epitopes formed by secreted proteins which contribute to the development of cancer and auto-immune diseases, including circulating tumor dervied soluble factors or inflammatory proteins. Typical examples are extracellular signalling molecules such as hormones, neurotransmitters, cytokines, growth factors or cell recognition molecules, cpable of attaching to the corresponding reeceptor and triggering changes in the funciton of the cell.STAGs include for example VEGF, RANKL, EGF, APRIL, TWEAT, TNF-alpha, OX40L, FasL, CD40L, IFN-alpha, IL-1 beta, IL-6, IL-13, GM-CSF, and TRAIL. 

Particular Bi-Specific Antibody Formats:

Fusions at N or C terminus:

Zhang (WO 2017/034770) discloses that the C terminus of a first CH3 containing polypeptide mayb be fused with a different H chain Fab region (VH and CH1), a ScFv, a probody, monobody, diabody, nanobdy, ligand or receptor or any kd of binding domains. A peptide linker may be used in between the fused binding unit and the first polypeptide chain. At the same time, the N-terminus of the second CH3 domain polypeptide maybe be fused with yet another diffferent H chain Fab region (VH and CH1), a ScFv, a probody, monobody, diabody, nanobody, lignd or receptor or any kind of binding comains. The C-tmerius of said seocnd CH3 comtaining polyeptide maybb be fused with yet another fidffernet H chain Fab region. 

Single-chain Fv (scFV):

The scFv format is the most commonly used derviative of the VH and VL domains representing the minimal antigen-binding site of an antibody. Due to the single-chain configuaiton, bisepcifc antibodies can be build by connecting tow scFvs thorugh a linker. Thus, these molecuels are bivalent with one valency for each antigen, with a typically size in the range of 50-60 kDa. The first descriptions of such tandem scFv molecules date back more than 20 years. In these studies, two scFvs were either fused by a 27 amino acid helicl linker or a flexible linker. Since then many other tandem scFv molecules have been geenrated. In principle, the format of taFvs are defined by the arrangement of the VH and VL in the two individual scFvs (i.e., VH-VL or VL-VH) and the lenght and composition of the connecting linker, which can affect correct folding, stability and antigen-binding of the molecule. Various connecitng linkers have been utilized, such as short alanine linkers, hydrophilic linkers and linkers derived form various immunogloublin and non-immunoglobulin molecules. (Brinkman “The making of bispecific antibodies, mAbs, 9:2, 182-212). 

–ScFv-CH3 and ScFv-Fc Fusion Proteins:

It may be desirable to produce antibody-based molecules larger than scFv.  A popular approach has been to fuse scFv to the hinge and Fc regions (scFv-Fv) to to just the thrid constant domain of IgG (scFv-CH3) without or without a hinge region. Fusion of scFv to Fc regions or CH3 domains results in proteins that spontaneously dimerize and can exhibit bivalent binding. Fc fusion generally retain the blood persistence properties of parental intact antibodies, whereas CH3 fusion proteins have clearance properties intermediate between scFv and larger proteins. The serum half life is most likely modulated by interactions between the Fc domains with the FcRn/FcRp receptor that controls serum persistence of immunoglobulins. Antibody-dependent cellular cytolysis (ADCC) and complement dependent cytoysis (CDC) can be linked to ovel antigen specificties using scFv-Fc fusions. (Su “ScV-CH3 and scFv-Fc fusion proteins” Antibody Engineering, pp. 646-652, 2001). 

Diabodies (Db): are bivalent molecules composed fo two chains, each comprising a VH and VL domain, either form the same or from different antibodies. In the diabody format, the two V domains are connected by a short linker that is usally 5 residues (e.g., GGGGS). Becasue the linker lenght is substantially shorter than that required to allow intrachain assembly of an antigen-binding site, whcih would result in a scFv, two chains dimerize in a ehad-to-tail orientation resulting in a compact molecule, with a molecular mass similar to tandem scFv (about 54 kDa). (Brinkman “The making of bispecific antibodies, mAbs, 9:2, 182-212)

Fc-less bispecific antibodies:

–Fab fusion protein: Fc less bispecific antibodies were obtained using fabs as the building block to which additional binding units are fused. Fabs are heterodimeric molecules composed of a L chain and a H chain fragment (Fd) and can thus be used to geenrate bivalent, bispecific molecules but also trivalent, bi- or ti specific fusion prtoeins (e.g., by fusing a scFv to the C-temrinus of either the L chain or Fd (bibody Fab-L-scFv, Fab-H-scFv0 or to both cahins (tribody, Fab-(scFv)2). (Brinkman “The making of bispecific antibodies, mAbs, 9:2, 182-212)

Methods of Generating and Identifying Variants

Useful Databa bases for Antibody Variant Design:  RCSB Protein Data Book (PDB) (provides structure and useful too in engineering design and data analysis). 

Articles/books:  Kuroda “Computer-aided antiboy design” Protein Engineering, Design & Selection (PEDS) 25(10: 507-21 (2012).  Nichols, “Rational design of viscosity reducing mutanants of a monoclonal antibody; hydrophobic versus electrostatic inter-molecular interactions, mAbs 7(1): 212-230 (2015); Talluri, “Advances in engineering of proteins for thermal stability” International J of Advanced Biotechnology and Research 1: 190-200 (2011); Prediction and Reduction of aggregation of monocloanl antibodies, J of Molecular Biology, 429(8): 1244-1261 (2017); Voynov “Predictive tools for stabilization of therapeutic proteins” mAbs 1(6): 580-582 (2009). 

The treatment of certain chronic ailments such as RA using mAb therapeis requires delivery via the s.c. route for pateints at home use. Tod eliver several hundres milligrams intot eh sc.c. space, a liquid formulation cotnaining high concentraitons of mAb is reuqired. Thus it is important that the lead clinical mAb candidate meets (a) an injectable solution of low viscosity (higher viscosity colutions are difficult to manufacture and adminsiter and could be painful to inject) (2) minimal chemical/physical degradation in solution and (3) a normal in vivo clearance provdie to vaoid multiple injections and/or more frequent dosing. In silico screeing can aid in selection of mAb candidates with respect to viscosity, in vivo clearnce in Cynomolgus monkeys (a relevant preclinical model for human clearance), Trp oxidaiton and Asp isomerication. mAb differening in the CDR sequences exhibit a variety of viscosity concentraiton profies under similar conditions of shear rate. (Sharma “In silico selection of therapeutic antibodies for development: viscosity, clearance, and chemical stability” PNAS, 111(52), 2014). 

In Silico design variants –Affinity Chromatography –Mass Spectrometry

Clark (US Patent Applicaiton NO: 146/409,818, published as 20190346456) discloses a method of generating/selecting an antibody variant amino acid sequence of interest from a plurality of antibody variant amino acid sequences which includes the steps of preslecting a reference antibody (e.g., trastuzumab), designing variant sequences of the reference antibody (e.g., in silico), cloning and expressing the sequences, purifying tne antibody sequences using affinity chromatography (e.g., Protein A – MabSelect SuRe) and then determining the molecular weights of each of the different variant antibodies which are eluted form the affinity chromatogrpahy by Mass Spectrometry to identify the particular variants.