High Affinity Promotes Internalization of Engineered Antibodies Targeting FGFR1

Abstract

Fibroblast growth factor receptor 1 (FGFR1) is a plasma membrane protein that transmits signals from the extracellular environment, regulating cell homeostasis and function. Dysregulation of FGFR1 leads to the development of human cancers and noncancerous diseases. Numerous tumors overproduce FGFR1, making this receptor a perspective target for cancer therapies. Antibody-drug conjugates (ADCs) are highly potent and selective anticancer agents. ADCs are composed of antibodies (targeting factors) fused to highly cytotoxic drugs (warheads). The efficiency of ADC strategy largely depends on the internalization of cytotoxic conjugate into cancer cells. Here, we have studied an interplay between affinity of anti-FGFR1 antibodies and efficiency of their cellular uptake. We have developed a unique set of engineered anti-FGFR1 antibodies that bind the same epitope in the extracellular part of FGFR1, but with different affinities. We have demonstrated that these antibodies are effectively taken up by cancer cells in the FGFR1-dependent manner. Interestingly, we have found that efficiency, defined as rate and level of antibody internalization, largely depends on the affinity of engineered antibodies towards FGFR1, as high affinity antibody displays fastest internalization kinetics. Our data may facilitate design of therapeutically relevant targeting molecules for selective treatment of FGFR1 overproducing cancers.

FGFR1 clustering with engineered tetravalent antibody improves the efficiency and modifies the mechanism of receptor internalization

“…We have recently reported a phage display‐based selection of high‐affinity antibody fragments that selectively recognize epitopes within the D1 domain of the FGFR1 [25]. Furthermore, we have demonstrated that the bivalency and the high affinity promote internalization of FGFR1/engineered antibody complexes [5,6,49]. To study the impact of FGFR1 clustering into larger oligomeric structures on the receptor activity and intracellular trafficking, we generated tetravalent engineered antibody, T‐Fc, composed of two anti‐FGFR1 scFv fragments recognizing D1 domain of the receptor fused to the Fc fragment of human IgG1.…”

“…Tetravalent antibody‐mediated FGFR1 clustering largely improves the uptake efficiency, which may boost the selective delivery of drugs into FGFR1‐overproducing cancer cells. This may be partially due to the very high affinity of tetravalent antibody for FGFR1, a factor recently demonstrated by us to play a significant role in the effectiveness of anti‐FGFR1 antibody internalization [49]. The activity of distinct endocytic pathways can be altered depending on physiological conditions [60–62].…”

“…Fully glycosylated extracellular domains of FGFRs fused to the Fc fragment of human IgG1: FGFR1 IIIc (FGFR1‐Fc), FGFR2 IIIc (FGFR2‐Fc), FGFR3 IIIc (FGFR3‐Fc), and FGFR4 (FGFR4‐Fc), and the extracellular part of FGFR1 lacking the N‐terminal D1 domain (FGFR1 D2‐D3) were produced as described previously by our group [48]. FGFR1 GST‐D1 was expressed in E. coli BL21 CodonPlus (DE3)‐RIL (Agilent Technologies, Santa Clara, CA, USA) and purified with use of Glutathione Sepharose column as described previously [5,6,49].…”

Considerations for the Design of Antibody-Based Therapeutics

“…63 Affinity of the antibody to the receptor antigen may also affect the rate of ADC internalization, with one study showing faster internalization for ADCs with tighter Ag affinity. 64 While ADC-targeted receptors should internalize efficiently, they should also display minimal shedding from the membrane. Such antigen shedding may lead to accumulation of extracellular ADC-antigen immune complexes and an increase in systemic toxicity.…”

Immunotoxins: Targeted Toxin Delivery for Cancer Therapy

“…The hybridoma technology introduced by Kohler and Milstein in 1975 enabled the production of monoclonal antibodies (mAbs) in a large scale. 1 Fundamental knowledge gained on cancer antigens or receptors, the structures and mechanisms of mAbs as well as the achievements in recombinant DNA techniques and devel-opments in modern protein engineering trigger people's interest in developing novel antibody-based cancer therapies, including new mAb constructs 2,3 and antibody-cytotoxic agent conjugates, 4 including immunotoxins. 5,6 In the past 20 years, the development of cancer therapeutic drugs was mainly focused on the field of mAbs.…”