High affinity anti-TIM-3 and anti-KIR monoclonal antibodies cloned from healthy human individuals

<div><p>We report here the cloning of native high affinity anti-TIM-3 and anti-KIR IgG monoclonal antibodies (mAbs) from peripheral blood mononuclear cells (PBMC) of healthy human donors. The cells that express these mAbs are rare, present at a frequency of less than one per 10⁵ memory B-cells. Using our proprietary multiplexed screening and cloning technology CellSpot™ we assessed the presence of memory B-cells reactive to foreign and endogenous disease-associated antigens within the same individual. When comparing the frequencies of antigen-specific memory B-cells analyzed in over 20 screening campaigns, we found a strong correlation of the presence of anti-TIM-3 memory B-cells with memory B-cells expressing mAbs against three disease-associated antigens: (i) bacterial DNABII proteins that are a marker for Gram negative and Gram positive bacterial infections, (ii) hemagglutinin (HA) of influenza virus and (iii) the extracellular domain of anaplastic lymphoma kinase (ALK). One of the native anti-KIR mAbs has similar characteristics as lirilumab, an anti-KIR mAb derived from immunization of humanized transgenic mice that is in ongoing clinical trials. It is interesting to speculate that these native anti-TIM-3 and anti-KIR antibodies may function as natural regulatory antibodies, analogous to the pharmacological use in cancer treatment of engineered antibodies against the same targets. Further characterization studies are needed to define the mechanisms through which these native antibodies may function in healthy and disease conditions.</p></div

Native anti-KIR antibodies cloned from healthy human donors were tested for cross reactivity with various members of the KIR family.

<p>The midpoint of ELISA binding curve was used to estimate the affinity as biosensor values are unreliable for very high affinity mAbs [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0181464#pone.0181464.ref018" target="_blank">18</a>].</p

The mean frequency of antigen specific memory B-cells for various antigens using CellSpot.

<p>Antigens are listed in order of increasing mean frequency of reactive memory B-cells within positive healthy donors.</p

Bio-Layer Interferometry (ForteBio Octet) was used to determine the binding of TRL antibodies to the extracellular domain of TIM-3.

<p>TIM-3 binding sites of the four mAbs are in close proximity since binding of the high affinity TRL6061 precludes binding of the three other antibodies to TIM3. TRL308 is an isotype control.</p

Native anti-KIR antibodies cloned from healthy human donors.

<p>All TRL mAbs regardless of their original isotype were cloned and tested as IgG1 mAbs (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0181464#pone.0181464.s002" target="_blank">S2 Fig</a>).</p

Pearson correlation matrix.

<p>The data demonstrate a strong positive association among the frequencies of positive memory B-cells for TIM-3), and infectious disease agents (biofilm, influenza B) as well as tumor-associated antigen (ALK).</p

The mean frequency of antigen-specific memory B-cells per 10⁵ total memory B-cells for twelve different antigens from 27 different donors using CellSpot.

<p>The mean frequency of antigen-specific memory B-cells per 10⁵ total memory B-cells for twelve different antigens from 27 different donors using CellSpot.</p