________________________________
From: Antibody Discussion Forum <email obscured>> on behalf of
Bover,Laura <email obscured>>
Sent: Thursday, April 11, 2019 4:01 PM
To: <email obscured>
Subject: Re: [ABRF Ab Forum] [EXT] Re: Novel heterodimer receptor mAb
External Email - Use Caution
Hi Ed,
Let me tell you some of our approaches:
1) Transduce/transfect murine fibroblast with the construction (TCR,
HLA/peptide), any other ECD). Immunize with those cells and screen against
those and parental mock. Do Dry cells ELISA for screening that pretty match the
FACS and is less work. In our hands dry cells are 99 % similar to FACS data,
and we can test 20 ELISA plates. Cells have to be coated in suspension in PBS
100k cells per well and dry on the hood. You can save the plates frozen until
use. The background in serum titration is kind of high for parental mock, but
as soon as you see a difference you can proceed to fusion and you will find
positive clones.
2) immunize with the TCR or complex receptor, or HLA/peptide. Don’t need KLH if
it is human and big. Screen against same protein biotinylated in an ELISA plate
coated with SV. This gives you a 3D aspect of the target. Anyway we screened
2,000 to get a couple.
I hope this helps!
Laura
Laura Bover, PhD
Professor
Director Monoclonal Antibody Core Facility
University of Texas M.D.Anderson Cancer Center
Associate member of The University of Texas Graduate School of Biomedical
Sciences at Houston
Member of the Clinical Research Committee 2
Member of the Steering Committee of the Immunology Program GSBS
Immunology Department/ Genomics Medicine Department
7455 Fannin St Room 1SCR4.3015
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<email obscured>
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On 4/11/19, 12:45 PM, "Antibody Discussion Forum on behalf of Greenfield,
Edward A.,Ph.D." <email obscured> on behalf of
<email obscured>> wrote:
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Hi Frances - Thanks for the advice. I agree it is exactly like making TCR
mAb. The PI is reluctant to try new things. I suggested the subtractive
approach and cDNA but they are too far off what he is used to doing. I was
thinking of immunizing with cells expressing the intact receptor. I am trying
to get him to change the screening assay from FACS to a blocking assay, which
may be more reflective of the antibody titer we are looking for. Ed.
-----Original Message-----
From: Antibody Discussion Forum <email obscured>> On Behalf Of
Weis-Garcia, Frances/Sloan Kettering Institute
Sent: Thursday, April 11, 2019 1:30 PM
To: <email obscured>
Subject: Re: [ABRF Ab Forum] Novel heterodimer receptor mAb
External Email - Use Caution
I think you are playing numbers game ... if the mouse contains a specific
antibody at all. I view it like making a TCR specific antibody, which is not
trivial but can be found. In the past we tried with purified protein and
failed after 4 attempts / strategies.
This year we just succeeded against another TCR. The mice were immunized
with primary DCs engineered to express the target TCR. If you cannot do that,
might want to try genetic immunization so the only thing that is different is
the heterodimer. I have been told by people who used a gene gun approach to
drive a T cell response that the endogenous DCs express the target. We have
genetically good humoral responses with both the gene gun as well as high
pressure tail vein injection (inject 2 mls plasmid in Ringers in 5 - 10
seconds) techniques ... so I would try this.
Have you tried subtractive immunization? Subtracting with cells expressing
one or the other and then driving the response you want with cells expressing
both.
If you try either of these approaches, it would be great if you could share
with group.
Frances
-----Original Message-----
From: Antibody Discussion Forum <email obscured>> on behalf of
Edward Greenfield <email obscured>>
Reply-To: <email obscured>>
Date: Thursday, April 11, 2019 at 1:06 PM
To: <email obscured>" <email obscured>>
Subject: [ABRF Ab Forum] Novel heterodimer receptor mAb
Has anyone had any experience making antibodies specific for a
heterodimeric receptors? The target receptor is composed of 2 different
chains, It is a novel receptor so antibodies are not available to either chain
individually. The desired antibody needs to be specific for the complete
receptor and not recognize either chain individually. We have immunized double
knock-out mice (for both chains) and have been able to get a titer of 1:8000
but cannot separate the individual responses from the intact receptor response.
Anyone have any suggestions for immunizing or screening? Ed.
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