First studies on tumor associated carbonic anhydrases IX and XII monoclonal antibodies conjugated to small molecule inhibitors

Figures & data

Scheme 1. General synthetic procedure for Mab-CA IX/XII conjugates reported in this study.

Scheme 2. Synthetic approach to CAI-functionalized peptides 1–7.

Table 1. RP-HPLC ESI-MS data for CAI-functionalized peptides 1–7.

Peptides*	Retention time (min)	[M + H]^+ calculated	[M + H]^+ found
1	13.97	821.30	821.02
2	14.04	793.27	792.81
3	13.01	793.27	792.81
4	11.35	801.27	801.1
5	11.37	801.27	801.1
6	14.67	803.27	803.04
7	16.90	858.27	858.2

*HPLC method: gradient 5%–50% B in 20 min, flow 1 ml/min. A (0.1% TFA in H₂O) B (0.1% TFA in ACN).

Table 2. hCA I, II, IX and XII inhibition data with MAb-CAIX/XII-CAI conjugates using the Acetazolamide (AAZ) as standard by a stopped flow CO₂ hydrase assay¹⁸.

Conjugate	KI (nM)*
	hCA I	hCA II	hCA IX	hCA XII
1) MAb-CA IX-1	>50 µM	>50 µM	0.04	>50 µM
2) MAb-CA IX-2	>50 µM	>50 µM	0.03	>50 µM
3) MAb-CA IX-3	>50 µM	>50 µM	0.06	>50 µM
4) MAb-CA IX-4	>50 µM	0.48	0.02	>50 µM
5) MAb-CA IX-5	>50 µM	>50 µM	0.05	>50 µM
6) MAb-CA IX-6	>50 µM	>50 µM	0.06	>50 µM
7) MAb-CA IX-7	>50 µM	>50 µM	0.09	>50 µM
8) MAb-CA XII-1	>50 µM	>50 µM	>50 µM	0.70
9) MAb-CA XII-2	>50 µM	>50 µM	>50 µM	0.55
10) MAb-CA XII-3	>50 µM	>50 µM	>50 µM	0.61
11) MAb-CA XII-4	>50 µM	15.1	>50 µM	0.08
12) MAb-CA XII-5	>50 µM	>50 µM	>50 µM	0.80
13) MAb-CA XII-6	>50 µM	>50 µM	>50 µM	0.79
14) MAb-CA XII-7	>50 µM	>50 µM	>50 µM	0.77
15) MAb-CA IX	nt	>50 µM	0.3	nt
16) MAb-CA XII	>10 µM [15]	>10 µM [15]	640 [15]	3.1[15]
AAZ	250	12	25	5.6

*Mean from 3 different assays, by a stopped flow technique¹ (errors were in the range of ±5%–10% of the reported values). Abbreviations: nt: not tested.

Khalifah RG. The carbon dioxide hydration activity of carbonic anhydrase. I. Stop-flow kinetic studies on the native human isoenzymes B and C. J Biol Chem 1971;246:2561–73.

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