Avid Bioservices Inc (CDMO)

[jazzbeerman]

catdaddy, Sure-




from one non-biologist to another...




bavituximab is an antibody, whose two "feet" (antigen binding regions) each need to grab one molecule of B2GPI in order to stick to PS.

Bavituximab, (like many antiphospholipid antibodies found in people), needs a cofactor to stick to its target phospholipid.

The feet of bavi stick to "domain 2" of B2GPI.
B2GPI's "domain 5" is actually what sticks to PS, - ESPECIALLY when you put together TWO B2GPI molecules - (which is what bavi does).

(Bavituximab is a "beta-2-glycoprotein-I-dependent anti-phosphatidylserine antibody)

Here's a simplified pic -







Now, after that was all understood, (which took some time, - please read paragraphs 233-255 of the most recent Thorpe/King/Luster patent application, which I will paste at the bottom of this post. It's a great recap of the insights and discoveries that led to the current understanding of bavi and why it is not one of the 'bad ones') -


Anyway, after that was all understood, ideas for 2nd generation drugs became obvious, hence- BETABODIES.....


A "Betabody" is a kind of "pre-made" complex of bavi+ two B2GPI's. You see, when bavi gets put into your bloodstream, each bavi molecule needs to grab TWO B2GPI's in order to form that particular complex that is so attracted to PS. There is a finite amount of available B2GPI at any given time. There is a curve of diminishing returns that comes into play, - if too many bavi molecules were shot into you, a higher percentage of them would only find ONE B2GPI molecule, resulting in a bunch of bavi's floating around that can't find a second B2GPI, which is a waste...



(Obviously one reason for all this intellectual property is to cover all the bases, the possible other ways to come up with recombinant molecules that do what bavi does)....


Anyway, the idea obviously came about of making that special complex in advance. If you can, (and you can), cut the two feet of an antibody off, and then glue two B2GPI molecules on where the feet used to be, you'd have that "bavi/two-B2GPI complex" all set up to go, in advance, and taking that bell-curve of PS binding out of play.





FURTHERMORE.............
If you'll read the most recent patent application,
http://appft1.uspto.gov/netacgi/nph-Parser?Sect1=PTO2&Sect2=HITOFF&u=%2Fnetahtml%2FPTO%2Fsea...
you'll see how important it is for PPHM to have gotten the rights for nicked B2GPI, in as much as it looks like another next generation drug may simply be two of the B2GPI's themselves linked together, or nicked bits or two of the PS-binding regions of B2GPI linked together, or any of who knows how many other possible combinations of various domains of B2GPI that can be designed to stick well to PS...


FOR INSTANCE - in the recent patent application, there's info on:
An artificial dimeric .beta.2GPI construct binds endothelial cells with exposed PS.....


The patent application, (the 'betabody' patent application) is a great read if you're interested. It would help folks see the connections/logic/progress between bavi, bit's of bavi,B2GPI, & various cleaved/nicked forms of B2GPI and all the possible combinations...




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bavi safety b2GPI safety, (differentiation from pathogenic autoantibodies) -




[0233] As with the other antibodies used herein, 3G4 therapy is well-tolerated in tumor-bearing animals treated repeatedly with the therapeutic dose (4 mg/kg in mice, three times a week). The mice retained normal physical signs, coagulation parameters, bone marrow cellularity, white blood cell counts and histology. Despite effects of very high concentrations of 3G4 in partially inhibiting phospholipid-dependent coagulation pathways, a substantial safety margin exists between the therapeutic dose and the dose that prolongs coagulation times in vivo. 3G4 is thus an effective and well-tolerated anti-tumor agent, which acts by homing to anionic phospholipids on tumor blood vessels and causing host cell-mediated antitumor effects. 3G4 is not associated with pathogenic effects reported in the literature for antibodies associated with anti-phospholipid syndrome(s).

[0234] Anti-phospholipid syndrome(s) (APS) are associated with autoantibodies termed "anti-cardiolipin" antibodies and "lupus anticoagulant antibodies". These syndromes are associated with a predisposition towards venous and arterial thromboemboli, thrombocytopenia and a number of neurological syndromes. The anti-phospholipid antibodies in these patients are thus "pathogenic antibodies". Such anti-phospholipid antibodies in the human population occur in systemic lupus erythematosus (Branch et al., 1987; Staub et al., 1989; Drouvalakis and Buchanan, 1998; Smimov et al., 1995; Rauch et al., 1986; Rauch and Janoff, 1990) and are associated with recurrent pregnancy loss (Rote et al., 1995; Rote, 1996; Vogt et al., 1996; 1997; Katsuragawa et al., 1997).

[0235] Although described for years as "anti-phospholipid antibodies" and "anti-PS antibodies", such pathogenic antibodies in fact recognize protein cofactors that bind to cardiolipin, PS or both, not the phospholipids themselves (Galli et al., 1990; 1993; McNeil et al., 1990; Rote, 1996). There is considerable heterogeneity in the pathogenic antibodies. Certain anti-cardiolipin antibodies have been reported to recognize particular regions on .beta.2-glycoprotein I, whereas lupus anticoagulant antibodies recognize prothrombin. Similarly, anti-PE antibodies that occur in disease states bind to PE in combination with proteins, such as low and high molecular weight kininogen (HK), prekallikrein and factor XI (Sugi and McIntyre, 1995; 1996a; 1996b).

[0236] In selecting antibodies for administration as therapeutics, it was thus thought that such antibodies should be identified on the basis of not binding to phosphatidylserine in combination with protein cofactors, but rather "true" anti-phosphatidylserine antibodies should be sought (WO 2004/006847).

[0237] However, further in vitro studies of the 3G4 antibody revealed that, unlike 9D2, binding of 3G4 to phosphatidylserine and anionic phospholipids was partially inhibited in the complete absence of serum. Moreover, binding was found to be restored when .beta.2-glycoprotein I (.beta.2GPI) was added. This prompted the inventors to believe that 3G4 recognizes an epitope in lipid-.beta.2GPI complexes, such that the interaction between 3G4 and PS is dependent on .beta.2GPI (U.S. provisional application Serial No. 60/646,333, filed Jan. 24, 2005; Ran et al., 2005). The inventors reasoned that PS exposed on tumor vessels in vivo is probably complexed with serum components, such as .beta.2GPI, and that 3G4 probably binds to these complexes.

[0238] The potential for 3G4 to bind to a PS-.beta.2GPI complex was very surprising, not least because 3G4 has been shown to be safe when administered to animals in numerous studies, and not to be associated with pathogenic effects reported in the literature for antibodies associated with APS, which antibodies are known to bind to lipid-serum protein complexes, including PS-.beta.2GPI complexes. No manifestations of APS have been observed in any 3G4 treatment, in contrast to those observed with anticardiolipin antibodies against .beta.2GPI (Matzinger, 1998; Fadok et al., 1998; Fadok et al., 2001a;b). Mice treated with 3G4 at high doses for prolonged periods showed no changes in coagulation capability, yet mice respond with APS when injected with anticardiolipin or lupus anticoagulant antibodies.

[0239] The present invention resolves this discrepancy, elucidates the interaction of 3G4 and .beta.2GPI required for binding to endothelial cells with exposed PS, and explains how 3G4 can bind to a PS-.beta.2GPI complex without succumbing to the toxicities associated with previously known pathogenic antibodies.

[0240] As shown in Example XXX, the interaction between 3G4 and PS is dependent on .beta.2GPI. Although .beta.2GPI binds anionic phospholipids weakly under physiological conditions, the present invention shows that 3G4 greatly enhances the binding of .beta.2GPI to PS-positive endothelial cells. The data show that divalent 3G4/.beta.2GPI complexes are required for enhanced binding, since 3G4 Fab' fragments do not bind endothelial cells with exposed PS. It is also demonstrated that an artificial dimeric .beta.2GPI construct binds to endothelial cells with exposed PS without the need for 3G4. Together, these data suggest that 3G4 targets PS-positive cells, including tumor endothelial cells, by increasing the affinity of .beta.2GPI for PS via the formation of a divalent 3G4/.beta.2GPI complex.

[0241] Example XXX also shows that 3G4 binds to domain II of .beta.2GPI. This is important, as antibodies from APS patients that recognize .beta.2GPI domain II are not pathogenic. In contrast, a significant recent study demonstrated that pathogenic anti-.beta.2GPI antibodies isolated from patients with APS commonly recognize domain I of .beta.2GPI (de Laat et al., 2005a). The ability of the 3G4 antibody to bind PS-.beta.2GPI complexes without binding .beta.2GPI domain I is important in the lack of toxicity that the antibody exhibits. .beta.2GPI can also interact with the apolipoprotein E receptor 2' (apoER2') (Lutters et al., 2003). Another recent study indicated that domain V of .beta.2GPI interacts with apoER2' and is involved in the activation of platelets, which causes increased platelet deposition to collagen (van Lummel et al., 2005).

[0242] Accordingly, by binding neither domain I nor domain V, the 3G4 antibody can bind to a PS-.beta.2GPI complex and yet show no toxicity following extensive toxicological studies performed in a variety of animal models. In light of these findings, other antibodies that bind to PS-.beta.2GPI complexes can now be made and used safely in the treatment of various conditions, such as cancer and viral infections. The selection of antibodies that do not significantly bind to .beta.2GPI domain I is currently the primary requirement, and the selection of antibodies that do not significantly bind to .beta.2GPI domain I or to .beta.2GPI domain V is envisioned to provide additional advantages.

[0243] In review, the new studies have characterized the interaction between the 3G4 antibody and its main anionic phospholipid target, phosphatidylserine. The data demonstrate that the interaction between 3G4 and PS is serum-dependent. .beta.2GPI was identified as the serum factor required to mediate the 3G4-PS interaction.






http://appft1.uspto.gov/netacgi/nph-Parser?Sect1=PTO2&Sect2=HITOFF&u=%2Fnetahtml%2FPTO%2Fsea...



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